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Synthesis of a molecularly tethered dual function germinant and antimicrobial agent.
Matthew Justin Hird
Doctor of Philosophy
Aston University
March 2014
©Matthew Justin Hird, 2014
©Matthew Justin Hird, 2014 asserts his moral right to be identified as the author of this thesis
This copy of the thesis has been supplied on condition that anyone who consults it is understood to
recognise that its copyright rests with its author and that no quotation from the thesis and no
information derived from it may be published without appropriate permission or acknowledgement.
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Aston University
Synthesis of a molecularly tethered dual function germinant and antimicrobial agent.
Matthew Justin Hird
PhD
2014
Clostridium difficile (C. difficile) bacteria are the leading cause of nosocomial diarrhoea in the UK.
The problem with the removal of C. difficile from hospitals is that it can sporulate and therefore be
difficult to remove/kill using conventional methods. The spores enter the body via the faecal-oral route
and in the presence of germinants (taurocholate), germinate into vegetative cells in the intestine, cause
infection and produce symptoms via the release of two main toxins.
The project’s aim was to produce polymeric steroid-based antimicrobial materials which will be able
to germinate spores and then destroy the resulting vegetative cells. Deoxycholic acid, lithocholic acid
and cholic acid were chemically manipulated to do this.
Various methods were tried to attach di-amines with varying tertiary amine-based groups to the parent
bile acids, with success found using ethyl chloroformate to activate the carboxylic acid group via an
anhydride group, with yields up to 90 %.
Once synthesised, the bile amides were screened for germinatory activity. The variables included the
chain length and the nature of the groups on the tertiary amine. Once germination had been achieved
the tertiary amine group was quaternized using various alkyl halides to introduce potential
antimicrobial functionality.
From the manipulation of the tertiary amine, several compounds were found to be germinants. Several
quaternized materials also displayed antimicrobial activity. Work was undertaken to attach acryloyl
groups to the 3-OH group of chemically modified lithocholic acid, with success of then polymerising
the monomer.
Co-germinants are amino acids, such as glycine and alanine which assist taurocholate in the
germination of C. difficile spores. It was therefore attempted to produce a polymerizable glycine
analogue which could be incorporated into the steroidal polymer to produce a germinatory surface.
Boc-Lys-OH was converted to its acrylamide derivative with a view to the incorporation of a tethered
glycine equivalent into a steroid polymer.
Key words; Clostrdium difficile, polymer, co-germinants, bile acids, nosocomial.
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Acknowledgements
Thanks go to my supervisor, Dr D Rathbone for his help and support throughout these three years. My
thanks go to Dr T Worthington, for helping with the testing of the synthesised compounds and
microbiology background. Thanks to Dr Mike Davis for running the laboratory. Thanks also go to
Amber Lavender, Christian Lowden and Kristian Poole, for their testing of my compounds.
Thanks also go to EPSRC and insight health for the funding to enable me to do this work, as well as
the EPSRC mass spectrometry facility at Swansea University for running my mass spectrometry
analysis.
Thanks go to my colleagues in my office, Pranav Bhujbal, Alex Quayle, Shibu Ratnayake and Baptiste
Villemagne.
Special thanks go to my parents, Michael and Mary Hird, who have provided me with the opportunity
to reach my goal of becoming a doctor and been very supportive to me throughout my life, for this I
am forever grateful.
On a separate note, I am also grateful to all my friends who work at Aston University; you have made
this a very special experience.
This thesis is dedicated to my parents.
“Research is what I’m doing when I don’t know what I’m doing”.
Wernher von Braun.
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Table of contents
List of figures - 7
List of Schemes - 10
List of tables – 12
Abbreviations - 13
1.0 Introduction - 14
1.1.1 Clostridium difficile - 14
1.1.2 Sporulation of Clostridium difficile - 16
1.1.3 Germination of Clostridium difficile cells - 17
1.1.4 NAP1/BI/027, the hyper-virulent form of C. difficile - 18
1.1.5 Toxins and symptoms produced from Clostridium difficile/CDI - 19
1.1.6 Current treatments for Clostridium difficile infection - 20
1.2.1 Bile acids introduction - 24
1.2.2 Germinants and inhibitors of Clostridium difficile spores - 29
1.2.3 Amides overview - 37
1.2.4 Amide bond formation - 38
1.2.5 Aminolysis reaction - 40
1.2.6 Carbonyldiimidazole - 40
1.2.7 DMT-MM - 41
1.2.8 Ethyl Chloroformate- 42
1.3.0 Co-germinants of Clostridium difficile spores - 42
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1.4.1 Protecting groups for bile acids - 45
1.5.1 Stereochemistry of bile acids - 48
1.6.1 Polymer overview - 48
1.6.2 Bile acid derived polymers - 50
1.6.3 Antimicrobial polymers - 51
1.7.1 Benzophenone introduction - 53
1.8.1 Quaternizable materials/antimicrobials - 57
1.8.2 Antimicrobial bile amides - 59
2.0 Experimental - 62
2.1.1 Instrumentation - 62
2.2.1 Ester preparation - 64
2.3.1 Amide formation - 68
2.3.2 Manipulations of 3-OH on bile acid derivatives - 122
2.3.3 Quaternization of tertiary amines in bile amide derivatives – 126
2.3.4 Amino acid analogue synthesis - 154
2.3.5 Benzophenone derivatives - 156
2.4.1 Germination efficacy of sodium taurocholate solution/alternative – 159
2.5.1 Unsuccessful Benzophenone reactions - 160
3.0 Results and discussion - 162
3.1.1 Overview of project - 162
3.2.1 Bile amide analysis - 164
3.3.1 Solubility and reactivity of bile acids - 169
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3.4.1 Preparation of bile acid esters - 171
3.5.1 Aminolysis of bile acid esters, successes and failures - 172
3.6.1 Aminolysis of bile acid esters - 176
3.6.1 Aminolysis of bile acid esters - 176
3.7.1 Activation of bile acids and attempted amine coupling - 178
3.8.1 Inverse addition amide formation reactions - 188
3.9.1 Attachment of pendant groups on 3-OH - 191
3.10.1 Ether linkage - 193
3.11.2 Ester linkage - 194
3.12.1 Polymeric compounds - 195
3.13.1 Quaternization of materials to induce antimicrobial activity - 197
3.14.1 Synthesis of amino acid analogues - 206
3.15.1 Bile amides containing fluorescent side chains-anthranilamide - 211
3.16.1 Benzophenone-based materials - 213
4.1.1 Germination results – 216
4.1.2 Overall trends in germination - 235
5.1.1 Future work - 236
6.1.1 Conclusions - 237
7.1.1 References - 239
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List of figures
Fig 1; Key steps in the sporulation of Clostridium difficile, modified from (Leggett et al., 2012),
squiggly line represents cell DNA – 17.
Fig 2: Two current treatments for C. difficile, vancomycin (compound 1) and metronidazole
(compound 2) (Tsutsumi et al., 2014) - 21
Fig 3: Structure of nitazoxanide (compound 3) and fidaxomicin (compound 5), new and emerging
antibacterial drugs used in the treatment of Clostridium difficile (Zhang et al., 2012) - 23
Fig 4: Diarylacylhydrazones (compound 3), a recently approved antibiotic in the treatment of C.
difficile - 24
Fig 5: Key bile acids, cholic acid (compound 6), deoxycholic acid (compound 7), lithocholic acid
(compound 8), chenodeoxycholate (compound 9) - 24
Fig 6: Naming and numbering system of the steroidal rings (compound 6) - 25
Fig 7: 3D structure of cholic acid, showing the curvature of the steroid structure (compound 10) - 26
Fig 8: A steroid based drug which helps to lower the MIC of other antibiotics (Savage and Li, 2004)
(compound 11) - 27
Fig 9: Structure of progesterone (compound 12), an inhibitor of C. difficile germination (Liggins et al.,
2011) - 29
Fig 10: Structure of pregnolone (compound 13) and dehydroepiandrosterone (compound 14), two
inhibitors of C. difficile spore germination - 30
Fig 11: Methyl chenodeoxycholate (compound 37) - 36
Fig 12: Taurocholate (compound 15), a compound usually conjugated with sodium in the body - 36
Fig 13: Resonance forms of an amide bond - 38
Fig 14: Showing the positions of the hydroxyls (compound 10) - 46
Fig 15: Inversion of stereochemistry at the 7 position in methyl cholate, (compound 48) - 48
Fig 16: A polymerized bile acid (compound 49) - 50
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Fig 17: Polymethracrylated cholic acid (compound 51) – 51
Fig 18; Polyethyleneimines, an antimicrobial cationic polymer (compound 51) - 52
Fig 19: PDMAEMA (poly (N, N-dimethylaminoethyl methacrylate, compound 52), an antimicrobial
polymer (Lu et al., 2007) - 53
Fig 20: Structure of benzopinacol (compound 53) and isobenzopinacol (compound 54), two products
from photo initiation of benzophenone - 56
Fig 21: An antimicrobial polymer, with quaternary ammonium ions, used in the prevention of S.
aureus. Modified from (Tiller et al., 2001) (compound 55) - 58
Fig 22: Squalamine, a steroid based antibiotic, effective against gram negative rods and gram positive
cocci (compound 56) (Aher et al., 2009) – 60
Fig 23: Lithocholyl-N-2-(2-aminoethyl) amide (compound 57), a bile amide which has shown in vitro
activity against gram positive bacteria (Ahonen et al., 2010) - 60
Fig 24: The carbon numbering system for a steroid based structure (in this case cholic acid). This
numbering system will be used in the bile acid derived compounds reported in this thesis - 63
Fig 25: PEEK polymer (compound 131) - 160
Fig 26: Proton NMR spectrum of lithocholic acid (top) and compound 102 (bottom). The horizontal
axis is in ppm – 166
Fig 27: Numbered carbons for compound 60 - 167
Fig 28: Chenodeoxycholic acid (compound 132) - 170
Fig 29: Two similar bile ester products, with very different yields, deoxycholic acid derivative
(compound 61) and lithocholic acid derivative (compound 60) - 171
Fig 28: Mixed anhydride of lithocholic acid with an unwanted carbonate formation on the 3-OH
position, due to excess ethyl chloroformate (compound 133) – 188
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Fig 29: Compound 87, an excellent germinator of C. difficile spores – 188
Fig 30: Possible dimerization formation from mixed anhydride induced inverse addition reactions
(compound 134) – 189
Fig 31: A potential polymerised co-germinant, based on L-lysine (compound 135) - 195
Fig 32: Compound 121, a quaternized polymeric compound of deoxycholic acid - 203
Fig 33; Compound 120, a potentially antimicrobial steroidal based polymer- 205
Fig 34: Compound 72, an aniline derived bile amide - 205
Fig 35: A bile amide analogue with 2-aminobenzamide (compound 84) – 212
Fig 36; Compounds 129 and 130, used in the potential production of benzophenone based surfaces -
214
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List of Schemes
Scheme 1: Proposed pathway for 7-dehydroxylation of cholic acid by liver biosynthetic enzymes
(Stellwag and Hylemon, 1979) - 28
Scheme 2: Carbodiimide induced amide formation mechanism (McMurry, 2004) - 39
Scheme 3: Aminolysis reaction (Talvik et al., 1999) - 40
Scheme 4: Formation of amide bond using carbonyldiimidazole - 41
Scheme 5: Amide bond formation using DMT-MM - 41
Scheme 6: Production of mixed anhydride using ethyl chloroformate - 42
Scheme 7: Mechanism of free radical polymerisation initiation by AIBN (Clayden, 2009) - 49
Scheme 8; Peroxy radical formation by allowing free radical polymerisation to occur in an oxygen
environment - 49
Scheme 9; Ground and excited state of benzophenone, with all possible outcomes upon exposure to
UV light- 55
Scheme 10: Overall synthesis plan for project - 162
Scheme 11: Esterification of lithocholic acid - 171
Scheme 12: Aminolysis between methyl lithocholate and an amine - 176
Scheme 13: Activation of lithocholic acid by means of ethyl chloroformate followed by amide
formation - 187
Scheme 14: Attachment of 4-vinyl benzyl chloride to methyl lithocholate - 193
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Scheme 15; Formation of an ester linkage at the 3-OH position on methyl lithocholate, using acryloyl
chloride - 194
Scheme 16: Compound 101, a methyl lithocholate which has had an acryloyl group attached at the 3-
OH position, which has then been polymerised – 196
Scheme 17: Quaternization of a tertiary amine by an alkylating agent - 203
Scheme 18: Synthesis method for a failed coupling between Z-Glu-OMe and 4-vinyl aniline - 208
Scheme 19: Attempted coupling of Boc-Ser-OH with vinyl benzyl chloride – 209
Scheme 20: Attempted coupling of methacrylic anhydride to Boc-Lys-OH -209
Scheme 21: Synthesis of manipulated amino acid (126) using a carbodiimide coupling agent - 210
Scheme 22: Compound 127; a monomeric polymerizable amino acid analogue - 210
Scheme 23: Mixed anhydride method of attaching an amino acid group to a bile acid - 211
Scheme 24: Synthesis method for a coupling between lithocholic acid and 4-amino-benzophenone -
213
Scheme 25: Binding of benzophenone based bile amides to organic surface containing C-H bonds -
213
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List of tables
Table 1: Structures tested by Howerton et al for germinating ability against C. difficile (Howerton et
al., 2010) - 31
Table 2: List of amino acids and small molecular weight compounds which act as co-germinants or
inhibitors of C. difficile spore germination - 43
Table 3: Key peaks of 13
C NMR in both reference material (far right column) and from product
(middle column) - 168
Table 4: Summary of all experiments tried using aminolysis methodology - 175
Table 5: Summary of all reactions attempted using coupling reagent methodology - 178
Table 6: All reactions attempted to manipulate the 3-OH moiety on lithocholic acid - 191
Table 7: All of the alkylation reactions which were attempted in this project - 202
Table 8: All the reactions attempted to make amino acid derivatives - 207
Table 9: Germination results for bile amide derivatives against C. difficile spore – 225
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Abbreviations:
AIBN - Azo-isobutyronitrile.
Boc- N-(tert-butyloxycarbonyl).
CDAD – Clostridium difficile associated disease.
CDI - Clostridium difficile infection.
C. diff – Clostridium difficile.
Ca2+
DPA – Calcium diplionic acid.
CDCl3 – Deuterated chloroform.
DCC – Dicyclohexyl carbodiimide.
DCM – Dichloromethane.
DMSO – Dimethylsulfoxide.
DMT-MM - 4-(4, 6-dimethoxy-1, 3, 5-triazin-2-yl)-4-methylmorpholinium.
MeOD – Deuterated methanol.
MIC – Minimum inhibitory concentration.
PDMAEMA - Poly (N, N-dimethylaminoethyl methacrylate).
THF – Tetrahydrofuran.
TLC – Thin layer chromatography
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Synthesis of a molecularly tethered dual function germinant and antimicrobial agent
Aim: To produce a polymer surface which is capable of germinating Clostridium difficile (C. difficile)
spores and killing the resultant vegetative bacterium.
1.0 Introduction:
1.1.1 Clostridium difficile:
Infections arising from pathogenic microorganisms are a significant concern in the world today,
especially in a hospital setting, with medical devices and surfaces being major reservoirs of infections
(Munoz-Bonilla and Fernandez-Garcia, 2012). These pathogens end up killing more people in the
world than any other single cause. C. difficile is a Gram positive, spore forming, obligate anaerobic,
rod shaped bacteria found asymptomatically in 3-15% of most healthy adult populations (Liggins et
al., 2011, Heeg et al., 2012) but more importantly is a nosocomial infection affecting both humans and
animals (Sorg and Sonenshein, 2010).
C. difficile was first discovered in 1935 by (Hall IC, 1935), while they were trying to understand the
development of natural gut flora in neonates. It wasn’t until 1978, however, that it was discovered that
the bacterium was responsible for pseudomembranous colitis (Badger et al., 2012). In humans, C.
difficile is found in the intestinal tract and it is also found in soil. In recent years, there has been a
sharp rise in cases of C. difficile infection, with numbers of fatal cases and incidences due to antibiotic
resistance increasing steadily, and the emergence of a hyper-virulent strain of C. difficile (Cecil, 2012).
Infections associated with the healthcare environment are widely regarded as the most frequent
adverse events of hospital life (Hook et al., 2012). C. difficile is one of the most important healthcare
associated infections in the UK, U.S.A and Canada (Koo et al., 2010). C. difficile associated disease
(CDAD) is the most usual cause of watery diarrhoea (Pyrek, 2013), colitis and pseudomembranous
colitis, due to the toxins produced by the bacterium. It is also the leading cause of diarrhoea in HIV
infected patients (Surawicz, 2000, Sorg and Sonenshein, 2008b, Howerton et al., 2010). There were
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over 14,000 reported cases of C. difficile in the financial 2012/2013 year in the United Kingdom and
in America where in 2000, they spent more than $1.1 billion on the treatment of the disease (Badger et
al., 2012). The incidence of the disease is rising and with 30% of sufferers not surviving the infection
(2010, Koo et al., 2010), this is a large morbidity count. It is only the vegetative form of C. difficile
which is able to produce the toxins which cause the symptoms (Howerton et al., 2010). Only the
metabolically active, vegetative bacteria can be killed by alcohol gels, not the spores which are able to
survive and resist many environmental stresses and are therefore, difficult to remove (Pyrek, 2013,
Leggett et al., 2012). As C. difficile is an obligate anaerobe, the vegetative cells are extremely
sensitive to oxygen, and if left in an aerobic environment will very quickly start to sporulate (Sorg and
Sonenshein, 2008a). Due to the spore’s resistance to normal cleaning measures, including alcohol
stations and the fact that it is they which are spread, they are known as the infectious stage of C.
difficile (Heeg et al., 2012). It is not known exactly how long the spores are able to survive for but
there has been the suggestion in the literature that they can survive for a very long time, some
literature suggests this can be many years (Kennedy et al., 1994).
One of the key problems with the diagnosis of the disease is that there is not one single identifying
assay that is sensitive, specific and rapid, thus delays, or false positive results are seen on a regular
basis (Hookman, 2009, McFarland, 2008).
C. difficile is predominantly found in hospitals, however, community infections are now also being
reported (Wilcox et al., 2008). Spread through the faecal-oral route, generally by healthcare
professionals not being as hygienic as they could be, the spores of C. difficile are ingested, and by their
nature they are able to resist the stomach acid. A major predisposing factor for CDAD (C. difficile
associated diarrhoea) is the overuse of broad range antibiotics (to which C. difficile is becoming
increasingly resistant, but natural gut flora is not) and as a result the natural normal gut bacterial flora
of the patient is significantly diminished. The natural gut flora would normally suppress the C. difficile
growth, but as it has been depleted, the vegetative cells are able to colonise the gut, grow unchecked
and produce toxins which can have severe symptoms. It has been shown that 25% of hospital cases of
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antibiotic associated diarrhoea is caused by C. difficile vegetative cells and that per gram of faeces,
approx. 100 spores are released (Wheeldon et al., 2008a).
1.1.2 Sporulation of Clostridium difficile.
When stressed (presence of air, lack of nutrients), C. difficile is able to form a spore which is very hard
to kill and difficult to remove from environmental surfaces. Harsh disinfectants including peracetic
acid or chlorine based disinfectants (such as chlorhexidine) (Fordtran, 2006) are the only materials
able to do this, but it would be impractical to wash a hospital in bleach or acid. It has been shown that
chlorine based disinfectants have sporicidal activity (Pyrek, 2013), but they are unpleasant to use and
have associated health and safety issues. In addition, bleach is corrosive and the use of these
disinfectants is therefore not ideal. Unfortunately, less harmful disinfectants such as 70% alcohol has
no effect on the spores of C. difficile, but it will kill vegetative cells.
There are several different, clinically relevant microorganisms which are able to sporulate. When
sporulation takes place it is usually categorised into seven unique stages illustrated in fig 1. At the start
of germination, the exosporium produces small filaments which are able to attach the whatever surface
the spore is on, thus preventing it being washed away (Sorg and Sonenshein, 2008a).
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Fig 1; Key steps in the sporulation of Clostridium difficile, modified from (Leggett et al., 2012),
squiggly line represents cell DNA.
1.1.3 Germination of Clostridium difficile cells.
When most bacterial spores (excluding C. difficile) germinate it is usually done through the Ger
receptor family of proteins. What distinguishes C. difficile spores from other bacteria is the lack of
these proteins. It is still assumed that the receptor sites in C. difficile spores are proteinaceous (Sorg
and Sonenshein, 2010), but no one is sure of how many different receptor sites there may be. At the
time of writing there is no known route of germination for these bacteria, only suggested hypothesis.
Germination is defined as, “the irreversible loss of spore-specific characteristics and ultimately leads
to vegetative cell growth”(Heeg et al., 2012). Germination can be observed in C. difficile spores
spectrophotometrically. When germination starts to occur there is a sudden decrease in the absorbance
at 600 nm. This indicates that the spore is going from phase-bright to phase-dark (when viewed using
a phase contrast microscope). Without spore germination, no colonisation of the gut occurs. If no
colonisation occurs, there is no release of toxins and therefore no infection. If the key factors of
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germination can be established, then it may be easier to stop germination occurring in the first place.
The presence of specific bile acids and co-germinants are the compounds which make the C. difficile
spores start to germinate, specifically (sodium) taurocholate (a secondary bile salt) and the amino acid
glycine. The mechanism of binding of each of the co-germinants is via a cooperative mechanism,
whereby the particular affinity of one co-germinant directly affects the binding of another (Howerton
et al., 2010).
The germination process has been fully studied in other spore forming bacteria such as Bacillus
subtilis. In these bacteria, germination is initiated by a proteinaceous germination receptor which is
encoded by a tricistronic operon. C. difficile does not have this receptor and therefore the exact
mechanism of germination is not completely understood, and with a scarcity of genetic tools available,
it will be this way for the foreseeable future (Howerton et al., 2010, Paredes-Sabja et al., 2008).
Although it is not known how it recognises germinants, the process that occurs after recognition is
well researched. Initially Ca2+
-dipicolinic acid is released, as well as H+, K
+ and Na
+ (Heeg et al.,
2012, Sorg and Sonenshein, 2010) which causes an uptake of water. This redistribution of ions causes
the lytic enzymes already present to become activated; these then degrade the spore cortex (a thick
peptidoglycan layer). The uptake of water ultimately degrades the cortex and therefore the outgrowth
of the bacterium can occur (Sorg and Sonenshein, 2010).
1.1.4 NAP1/BI/027, the hyper-virulent form of C. difficile.
Some literature suggests there has recently been an increase in number of infections due to the
emergence of the hyper- virulent strain of the bacterium known as NAP1/BI/027 (Costello et al., 2008)
(>35 % of all infections are caused by this strain). This strain has a higher antibacterial resistance
(specifically against fluoroquinones (Cecil, 2012)) and is associated with increased number of CDI (C.
difficile infection) which with it, has an increased mortality rate of 1-2.5 % (Liggins et al., 2011).
Some papers suggest the reason for this increased mortality rate is the ability of this strain to produce
toxins A and B quicker and in larger quantities (hyperproduction). The bacterium is able to do this due
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to a mutation in its tcdC gene (Badger et al., 2012), which also allows it to germinate at a higher rate
compared to other strains of the bacteria. Due to this mutation a rise in antibacterial resistance
(fluoroquinones, levofloxacin, and moxifloxacin) has also been found. The fact that C. difficile is
already very adept at going from a spore to a vegetative state (hypersporulation) means the spread of
this bacteria can be very quick indeed. (Heeg et al., 2012), strongly argue against this idea of a new
strain, however, and with no definitive review there is conflict on this particular strain in the literature.
1.1.5 Toxins and symptoms produced from Clostridium difficile/CDI.
Once the bacteria have colonised the small intestine (naturally occurs in 1-15% of healthy adults, 80 %
in neonates) (Badger et al., 2012)), they start to produce two known toxins, TcdA (enterotoxin, 308
kDa) and TcdB (cytotoxin, 270 kDa, more important in pathogenesis in animals(Sorg and Sonenshein,
2010)), with a possibility of a third toxin TcdC, about which little is known, other than perhaps a
synergistic relationship with the other two toxins and it being a binary toxin (Young and Hanna,
2014). TcdA causes cytotoxic loss of epithelial barrier function and specifically targets Rho GTPase
by being absorbed into the intestinal epithelial cells. This simply means that the junctions between the
epithelial cells are loosened and therefore destructive leukotrienes and cytokines are released and
cause damage (Hookman, 2009). Within minutes of exposure to TcdA there is considerable damage to
the mitochondria of the cells, the levels of ATP are greatly reduced and there is an abundance of
oxygen radicals which are likely to cause even more damage (Hookman, 2009). TcdB merely has a
synergistic relationship with toxin A and helps it to function. Thus, toxin A is the major virulence
factor. The other problem encountered with the toxins is that they are extremely unstable and are
degraded within 2 hours of being outside of the host’s body. Therefore, if a person is suspected of C.
difficile infection, it is imperative that they have their stool sample checked as soon as possible to see
if there is any toxin present in the stool sample. Failure to do so can cause false negatives to be found,
thus delaying of what could be critical treatment (Pyrek, 2013).
The most common effect of these toxins is colitis, but more severe symptoms such as toxic
megacolon, ileal perforation, fulminant colitis, brain empyema, colonic perforation, sepsis and
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colectomy have also been reported (Koo et al., 2010, Pyrek, 2013, Guo et al., 2012). The toxins bind
to the specific receptor on the colonocyte and this causes endocytosis of toxin-receptor complex into
the cell. Once the complex is inside the cell, it causes a drop in pH and a refolding of the toxin means
it is inserted into the trans membrane domain of the endosomal membrane. This is followed by a
translocation and release of catalytic domain into the cytosol via a mediated pore. Finally this causes
uridine diphosphatase-glucose dependent monoglycosylation of Rho-GTPase targets (Fordtran, 2006,
Hookman, 2009).
1.1.6 Current treatments for Clostridium difficile infection.
There are currently two main drug treatments for C. difficile infections, vancomycin (fig 2) which at
high doses has been shown to give patients more severe C. difficile diarrhoea (Surawicz, 2000) and
metronidazole (fig 2). Both of these drugs are becoming less effective, with recurrence rates being as
high as 20-25% (Mahony et al., 1999, Koo et al., 2010), with 50 % of those recurring sufferers going
on to relapse more than once (Badger et al., 2012). Although vancomycin has been shown to be the
better treatment for severe CDI, even with these drugs, there is up to a 20% relapse rate, which
requires a second round of treatment with antibiotics (Mahony et al., 1999). There has been a
suggestion that vancomycin is the better treatment but there have still been deaths even when it was
used as a treatment.
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Fig 2: Two current treatments for C. difficile, vancomycin (compound 1) and metronidazole
(compound 2) (Tsutsumi et al., 2014)
Metronidazole is a nitro aromatic prodrug which, when reduced on the 5 nitrogen becomes active
against the bacteria. It is a very low molecular weight drug and therefore it is easily transferred across
the cell membrane (Tsutsumi et al., 2014). Due to its relative low cost, compared to vancomycin, it is
usually the first prescribed antibiotic for the treatment of C. difficile associated disease.
Vancomycin is a hydrophilic (and therefore poorly absorbed in the GI tract), high molecular weight,
rigid glycopeptide antibiotic, consisting of a glycosylated hexapeptide chain and cross linked aromatic
rings by aryl ether bonds. Vancomycin works by tightly binding to the Ala-Ala sub-unit of the
precursor DP-N-acetylmuramylpentapeptide of peptidoglycan, which produces a complex through the
formation of hydrogen bonding. The resulting complex inhibits the biosynthesis of peptidoglycan, a
key component of the bacterial cell wall (Tsutsumi et al., 2014).
Other than the widespread overuse of antibiotics (all antibiotics have been implicated in this, other
than aminoglycosides, which do not cause CDI) (Williams and Spencer, 2009), there are several other
risk factors when it comes to the prevalence of C. difficile (specifically colitis) in hospitals. These
1
2
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factors include; advanced age (>65), gender (male) (Drekonja et al., 2011), impaired immune system
such as HIV/AIDS, malnutrition and post pyloric tube feeding (Fordtran, 2006, Badger et al., 2012).
There are some variants of C. difficile which don’t have the usual symptoms of diarrhoea, which can
make it particularly difficult to diagnose and therefore successfully treat. It has also been shown that
there are, in fact, specific antibiotics which can make the infection worse: penicillin, cephalosporin
and clindamycin being the main culprits, as well as other treatments such as glucocorticoids (Liggins
et al., 2011). Clindamycin was the first drug to be reported to produce severe diarrhoea when being
used to treat anaerobic infections (Tedesco, 1984).
There are several new antibiotics which are currently being tested as a treatment to C. difficile, such as
fidaxomicin (shown below, fig 3), the first drug to be approved for the treatment of C. difficile by the
F.D.A in over 20 years (Stranges et al., 2013). It is a poorly absorbed macrocycle which is highly
specific against the whole family of clostridiaceae. It works by inhibiting DNA transcription. The
main advantage this drug has over other drugs is that it has a cure rate which is similar to vancomycin,
but with reduced relapse rates and is still as effective at concentrations 4 times lower than both
vancomycin and metronidazole (Zhang et al., 2012, Lancaster and Matthews, 2012). Other drugs
currently being tested are nitazoxanide (fig 3), rifaximin, ramoplanin and tigecycline (Koo et al.,
2010). The problem with fidaxomicin is the fact that it is expensive compared with other treatments
($2500 per course) (Lancaster and Matthews, 2012) and its recurrence rates with the hyper-virulent
strain of C. difficile are the same as vancomycin and metronidazole (Chen et al., 2014).
Another school of thought is the use of probiotics, thereby replacing the natural gut flora which is
decreased by broad range antibiotics (Koo et al., 2010). However, a systematic review in 2008 by
Pillai and Nelson found there to be no significant evidence for the use of probiotics in treating of C.
difficile infection (Pillai A, 2008, Hookman, 2009), but in 2013 Schoster et al found there were at least
5 probiotic strains which would be complementary to the treatment of both Clostridium difficile and
Clostridium perfringens (Schoster et al., 2013), two of which are recommended to be taken forward
for further testing. In this area at least, there would appear to a lot of disagreement in the literature
- 23 -
with no definitive answer. One of the newest class of antibiotics, specifically acting against C. difficile
are the diarylacylhydrazones (fig 4) which have a protonophoric (prevention of movement of protons
across cell membranes) mechanism (Chen et al., 2014).
Further study is being done to develop a vaccine for the disease, by inducing an immune response to
the toxins, but this is still in early stages (Sougioultzis et al., 2005). There are also phase two trials
going on to see if, by introducing non-toxigenic C. difficile into the body, they will take up the same
niche as the toxigenic versions, therefore bringing the numbers of toxigenic bacteria down to a more
manageable level (Cecil, 2012). The only current successful treatment of C. difficile infection is the
use of faecal transplantation, which has a recorded success rate of 92 % (Drekonja et al., 2011), in
recurring infections.
Fig 3: Structure of nitazoxanide (compound 3) and fidaxomicin (compound 5), new and emerging
antibacterial drugs used in the treatment of Clostridium difficile (Zhang et al., 2012).
3 4
- 24 -
Fig 4: Diarylacylhydrazones (compound 3), a recently approved antibiotic in the treatment of C.
difficile.
1.2.1 Bile acids introduction:
Fig 5: Key bile acids, cholic acid (compound 6), deoxycholic acid (compound 7), lithocholic acid
(compound 8), chenodeoxycholic acid (compound 9).
Bile acids belong to the coprostane family (Zhu and Nichifor, 2002) and are based on steroidal
structures with several chiral centres. In nature they are biosynthesised from cholesterol (El Kihel et
al., 2008) in eukaryotic cells and are absent from the prokaryotes (Liggins et al., 2011). The reason
that there are so many closely related steroid structures is because there are so many different
biochemical pathways set up for the conversion of cholesterol into highly water soluble
membranolytic compounds (Hofmann and Hagey, 2008). They are used in a number of
6 7
8
9
5
- 25 -
pharmaceutical products, such as carriers for drugs which are specific to the liver and the dissolving of
gallstones (Kagedahl et al., 1997), due to their non-toxicity (Koivukorpi et al., 2007, Hu et al., 2005).
Various analogues of bile acids have exhibited antibacterial, antifungal (Tamminen and Kolehmainen,
2001) and antiviral activity as well as a metal complexing ability (Joachimiak et al., 2008). There are 4
saturated rings which are lettered in the steroidal backbone (fig 6) (Hofmann and Hagey, 2008), from
left to right, a-d (fig 6);
Work has established that the nature of the groups chemically attached to C-24 greatly dictates the
kind of properties that the compound has. If there is a hydrophobic component to it then they have a
tendency to be more active against gram negative bacteria (Guan et al., 2000). If there is a particularly
short chain, they are able to make the outer cell membrane permeable to other materials, and in fact
kill gram positive bacteria.
Fig 6: Naming and numbering system of the steroidal rings (compound 6).
The inherent skeletal structure of the bile acid with its four rings, covering an area of ~ 10 x 6 Å
(Davis and Joos, 2003) seems to be very important as there have not been any structures which
germinate the bacteria without this steroidal backbone. There would also appear to be a direct
correlation with increasing amounts of hydroxyl groups in the 3, 7 and 12 positions and germinating
ability i.e. cholic acid is a better germinator than lithocholic acid (Liggins et al., 2011).
6
- 26 -
Bile acids are an unusual class of molecules, due to their large, rigid but ultimately curved structure
(Gao and Dias, 1999, Zhu and Nichifor, 2002). Each of the hydroxyl groups in cholic acid is
chemically different in reactivity. Bile acids have a unique amphilicity and because of this can form
micelles very easily (Idziak et al., 1999, Hu et al., 2005, Zhu and Nichifor, 2002) and are known
emulsifiers (Zhang et al.). This is due to the positions of the methyl groups on the β face pointing “up”
due to being in the axial position while the hydroxyl groups on the α face all point “down”, due to the
equatorial position of 3-hydroxyl and the axial positions of 7 and 12 (in cholic acid, one of the most
common bile acids found) as shown in Fig 7. As there is a cis AB ring junction, this produces the
curvature of the structure. There are several structural variants with regard to the carbon chain length
attached to C20, with an addition of an extra four carbons being the most common in mammals (Fig
7). These are known as cholanes, where the four rings are all completely saturated. In the body these
structures are usually conjugated to the amino acids glycine or taurine (Hofmann and Hagey, 2008,
Zuluaga et al., 1999). Over the last couple of years there have been many modified bile acid drugs
produced, which have both anticancer and antibacterial activity (Huang et al., 2009). It has also been
shown that the introduction of steroid based drugs with antibiotics in the delivery system can lower the
MIC (minimum inhibitory concentration) of said antibiotic (Fig 8). The steroid based compounds are
able to do this by being inhibitors of the enzymes which are key in digestive processes in the liver and
thus increase the bioavailability of the potential drug molecules (Ahonen et al., 2010).
Fig 7: 3D structure of cholic acid, showing the curvature of the steroid structure (compound 10).
10
- 27 -
Fig 8: A steroid based drug which helps to lower the MIC of other antibiotics (Savage and Li, 2004)
(compound 11).
Lithocholic acid (3α-hydroxy-5β-cholan-24-oic acid) (8) and deoxycholic acid (3α, 12α-dihydroxy-5β-
cholan-24-oic acid) (7) are both secondary bile acids which are metabolised from primary bile acids
naturally via enteric bacteria modification of either chenodeoxycholic acid (9) or cholic acid (6) in the
liver, specifically a 7-α-dehydroxylation (by dehydroxylase enzymes) to form a double bond
intermediate at the 7-position, which is then hydrogenated to produce the deoxycholic acid (Scheme 1)
(Stellwag and Hylemon, 1979).
- 28 -
Scheme 1: Proposed pathway for 7-dehydroxylation of cholic acid by liver biosynthetic enzymes
(Stellwag and Hylemon, 1979).
- 29 -
1.2.2 Germinants and inhibitors of Clostridium difficile spores.
Fig 9: Structure of progesterone (compound 12), an inhibitor of C. difficile germination (Liggins et al.,
2011).
Progesterone (fig 9) has been shown to be an inhibitor of C. difficile spore germination, which means
it stops the spore from germinating into a vegetative cell. This shows that there is clearly some
interaction of the steroidal body with the bacteria, as there are no hydroxyls present in this compound,
indicating that the presence of hydroxyls in lithocholic, deoxycholic and cholic acid may have some
important function in promotion of germination. This has also been shown in other steroid based
materials such as corticosterone, which has a hydroxyl group in position 11 and has decreased
inhibitory effect compared to similar structures without the hydroxyl group present (Liggins et al.,
2011). Movement of the double bond (as in pregnenolone (13)) or exchanging the ketone group for a
sulfonic acid group (fig 10, compound 14) does not appear to make a difference; these materials are
still inhibitors of Clostridium difficile germination.
12
- 30 -
Fig 10: Structure of pregnenolone (compound 13) and dehydroepiandrosterone (compound 14), two
inhibitors of C. difficile spore germination.
Chenodeoxycholate (9) has been shown to be a germination inhibitor of certain strains of C. difficile
(Heeg et al., 2012). Unlike other materials which are germinants, it does not have a hydroxyl group at
position 12, suggesting that although the 12 hydroxyl is not important for the binding of a material to
C. difficile spores, it is in fact very important if the compound is to cause C. difficile spores to
germinate.
Chenodeoxycholate is a competitive inhibitor and therefore binds to C. difficile spore and prevents
other known germinating compounds such as sodium taurocholate from binding at the active site. This
suggests that the inhibitors most likely bind to the same place as the germinants do (Sorg and
Sonenshein, 2010, Heeg et al., 2012). However, there may be other allosteric receptor sites where it
may bind, thus changing the shape of the primary binding site, preventing germinants from attaching
to the binding site. Chenodeoxycholate is absorbed through the colonic epithelium where it is usually
metabolised to lithocholic acid, a poorly soluble bile acid with no germinating ability. When the gut
flora are diminished due to the overuse of antibiotics, the dehydroxylation does not occur very quickly
and chenodeoxycholate is absorbed 10 x quicker by the colonic epithelium than cholate (Sorg and
Sonenshein, 2010).
Howerton et al synthesised several different isomers of taurocholate to try and establish some structure
activity relationship. Below in table 1, is a summary of the compounds which were tested.
13 14
- 31 -
Table 1: Structures tested by Howerton et al for their germinating ability of C. difficile (Howerton et
al., 2010).
Compound
number
Structure % Germination of C. difficile
15
97.9 %
16
Reduction of 70 %
17
10 % of initial germination
ability
18
3 % of initial germination
ability
19
No germination
20
No germination
- 32 -
21
No germination
22
No germination
23
No germination
24
No germination
25
96.5 %
26
No germination
27
No germination
- 33 -
28
No germination
29
No germination
30
No germination
31
59.5 %
32
No germination
33
No germination
34
No germination
- 34 -
35
No germination
36
No germination.
Compound 15 is the structure for taurocholate. This has three hydroxyl groups in the 3, 7 and 12
positions. Taurocholate is a natural bile salt which is a very good germinant of C. difficile spores with
a 50 % effective concentration at 15.9 nM. In the body this compound is usually present as the sodium
salt and is the gold standard for germinating ability. The other compounds tested in Howerton et al are
all synthetic analogues of this compound. Previous work in this area had shown that sodium
taurocholate had significant solubility issues and thus was not appropriate for this project.
Compound 16 is lacking the hydroxyl at position 7. This removal has lowered its germinating ability
by 70 % (relative to taurocholate), thus showing that the 7-OH is very important in germination of C.
difficile spores. Compound 17 is similar to compound 16 but is missing the 12 hydroxyl instead of 7.
This hydroxyl is clearly more important as a germinatory factor as without it, as germination is only
10 % of taurocholate. Compound 18 is a further analogue of compound 17 but the 7 hydroxyl has been
changed from the alpha configuration to the beta configuration. By inverting the stereochemistry of
this substituent the germinating ability of the compound falls from 10 % to 3%, showing how
important the stereochemistry is.
Taurolithocholate (compound 19) lacks hydroxyls at both position 7 and 12, while taurocholanate
(compound 20), has no hydroxyl groups present at all. Unsurprisingly both of these compounds are
unable to induce any germinatory effects, thus showing the importance of these hydroxyl groups.
- 35 -
In compound 21 and compound 22 the 12 hydroxyl group has been moved to the 6 position on the
steroid backbone. By moving the hydroxyl group, this severely inhibited any germination of C.
difficile spores.
The Howerton group discovered that if taurocholate is O-methylated, two different compounds are
produced, 3-methoxy-7, 12-dihydroxytaurocholate (compound 23) and 3, 7-dimethoxy-12-
hydroxytaurocholate (compound 24). Neither of these compounds were able to induce or inhibit
germination of C. difficile spores.
Compounds 25- 36 were all variations on the taurine side chain to see if manipulation of this side
chain would have any effect on the germinatory ability of the compounds to germinate C. difficile
spores.
Compound 25 has one less carbon in its taurine side chain and this results in a marginal germination
decrease from 97.9 % for taurocholate to 96.5 %. Whilst removal of this carbon doesn’t hinder
germination, it doesn’t show any real inhibitory value either. However, addition of an extra carbon in
the chain, compound 26, negates any germinatory ability. This would suggest that the putative
germination receptor is relatively small so any expansion in size of the chain, or indeed, insertion of
extra groups, such as benzene rings or extra amide groups (compounds 27-30, 32-35), will leave the
compound completely unable to germinate C. difficile spores.
Compound 31 is similar to compound 25 but the change is conversion from a sulphonic acid group to
a carboxylic acid group. This conversion reduced germinating ability to 59.5 %, while conversion of
the amide of taurocholate to an ester removes any germinatory activity whatsoever.
Lithocholic acid has been shown to be an inhibitor of germination in some literature, even in the
presence of taurocholate. The reason it is not used as a medicine is because of its poor solubility and
the implication of its role in colorectal carcinogenesis (Sorg and Sonenshein, 2010). The 7 hydroxyl
could be very important when it comes to germinating, but not binding, much like deoxycholic acid.
Lithocholic acid does not have the 7-OH and is not a germinant. Deoxycholic acid is an inhibitor of
- 36 -
vegetative growth, where no growth was recorded at all (Paredes-Sabja et al., 2008, Wilson, 1983).
Cholic acid, which has all three hydroxyls present, has been shown to be a very good germinator of C.
difficile spores, although not as good as (sodium) taurocholate. The potential analogues of this
compound could prove to be very good at causing germination of the spores, converting them into
vegetative cells.
Fig 11: Methyl chenodeoxycholate (compound 37).
Taurocholate (fig 12) has been shown to be one of the most effective bile acid germinant of C. difficile
spores in a clinical setting, with or without the presence of co-germinants. It is so far the best
germinating material known, but extensive exposure to the material is required for germination to
occur (Wheeldon et al., 2008a).
Fig 12: Taurocholate (compound 15), a compound usually conjugated with sodium in the body.
37
15
- 37 -
(Sodium) taurocholate is a naturally occurring bile salt which has hydroxyl groups on the 3, 7 and 12
positions, respectively. If the hydroxyl group at position 7 is missing, the material is known as
taurodeoxycholate (16); this has been shown to germinate C. difficile spores as well, but at a rate 70%
less than standard (sodium) taurocholate. It would therefore appear that the key groups for germination
are the 7 and the 12 hydroxyls in the alpha position. However the hydrogen bonding ability of the 3-
OH is also important for binding and therefore germinating ability.
Removing the amide bond and replacing it with an ester in taurocholate negates any germinating
ability of the compound as well, thus showing that the amide group, and presumably therefore, the
group’s ability to form hydrogen bonds is very important to producing a germinatory response
(Howerton et al., 2010, Aher et al., 2009, Tiller et al., 2001).
Although there is a lot of data on the materials which will and will not cause germination of spores,
there are other factors which may be important to germination but which have not been fully
investigated at this point. These factors include but are not limited to pH, temperature and pressure.
Although inside the body all three of these are likely to be pretty stable, outside of the body, which is
the area this project is focussed, these factors can be highly variable (Wheeldon et al., 2008a).
1.2.3 Amides overview:
Amides are ubiquitous in nature and are found to exist in lots of complex synthetic and natural
compounds, such as proteins, used to join individual amino acids together, known as peptide bonds
(Allen et al., 2012, Allen et al., 2009). These bonds can be produced both via chemical and biological
means (Allen et al., 2010). However, there are difficulties with the synthesis of this bond, such as low
yield, racemisation, degradation and difficult purification (Montalbetti and Falque, 2005).
Amide bonds are strong because they are able to form different resonance forms of the bond. In nature
this bond can be formed using enzymes and therefore has minimal amount of waste material
associated with it, unlike modern day methods which often involve the use of inorganic catalysts or
ineficient reagents. This in turn makes the whole process environmentally unfriendly and costly. There
- 38 -
are many ways of synthesising an amide bond, with different reagents and conditions and yields. Often
what the amide is attached to will dictate the preferred method of amide coupling.
Fig 13: Resonance forms of an amide bond.
1.2.4 Amide bond formation
While the use of acid chlorides and p-nitrophenyl-esters have been proven to be the highest yielding
reagents, the execution of the synthesis can be difficult, whereas production of an anhydride, although
generally lower yielding, is a much simpler methodology (Bellini et al., 1990, Cravotto et al., 2005).
Anhydrides are species which will readily react with many different nucleophilic species, especially
alcohols to form esters and amines to form amides. The advantage ethyl chloroformate (an anhydride
forming reagent) has over DCC (dicyclohexyl carbodiimide); (the more traditional reactive species) is
that dicyclohexylurea is formed as a by-product, which can be difficult to remove. With anhydrides,
this is not the case and therefore, this has the advantage, especially if using an acid which is more
valuable (Montalbetti and Falque, 2005).
- 39 -
Scheme 2: Carbodiimide induced amide formation mechanism (McMurry, 2004).
There are several different types of carbodiimides, depending on the reaction type. DCC
(dicyclohexylcarbodiimide) as shown in scheme 2 produces a urea which is very poorly soluble and is
generally removed by filtration. This coupling method is therefore used mostly in solution phase
chemistry. If the requirement is to use solid phase chemistry, then DIC (diisopropylcarbodiimide) is
used as the resulting urea can be removed by DCM washes (Montalbetti and Falque, 2005).
Acid chlorides are very reactive species and will readily react with amines to produce amide bonds.
They are often introduced into the reaction with N, N-dimethylaminopyridine (DMAP) which behaves
as a catalyst. Pyridine can also be used as the solvent for many acid chloride reactions and produces
and intermediate acylpyridinium salt. Acid chlorides do have limitations however, they are easily
hydrolysable, can produce racemic mixtures of compounds and can potentially unwanted side
reactions. This potential for racemisation and side reactions to occur can sometimes be limited by the
- 40 -
use of acyl fluorides or acyl bromides, as they are less moisture sensitive and are more reactive
towards amines.
1.2.5 Aminolysis reaction
Aminolysis (scheme 3) is simply the heating up of a methyl ester of a bile acid with an amine, at a
temperature over 180 oC, producing methanol as a by-product. It has been shown that this reaction has
a rate limiting step which is the collapse of the tetrahedral intermediate. Unsurprisingly, the rate of
disappearance of the ester was equal to the rate of formation of the alcohol, in this case methanol
(Talvik et al., 1999).
Scheme 3: Aminolysis reaction (Talvik et al., 1999)
1.2.6 N, N’-Carbonyldiimidazole
There are many reagents which are useful in the coupling of a carboxylic acids and amines (not
specifically bile acid), as this is a key step in peptide chemistry. Literature suggests that the reagent
which was most likely to work on bile acids would be N, N’-carbonyldiimidazole (scheme 4)
(Larriv e-Aboussafy et al., 2009, Woodman et al., 2008). Carbonyldiimidazoles are generally
preferred to the use of acid chlorides because the intermediate acyl imidazole compounds are a lot
more stable (Larriv e-Aboussafy et al., 2009). The advantages of carbonyldiimidazole are the fact that
it is inexpensive. The only by-products produced are carbon dioxide and imidazole, which in relative
terms are reasonably benign, and therefore, if the reaction needed to be scaled up at some point, would
not be a problem (Woodman et al., 2008). The same paper also suggests that the use of imidazole. HCl
- 41 -
would improve the rate of reaction because of the presence of a proton donating compound,
protonating the imidazole, which would therefore make it more reactive.
Scheme 4: Formation of amide bond using N, N’-carbonyldiimidazole.
1.2.7 DMT-MM
Care must be taken when choosing the solvent system for the reaction. Alcohol-based solvents for
example, are likely to form the corresponding esters and therefore not appropriate. One paper has
reported a way around this by using 4-(4, 6-dimethoxy-1, 3, 5-triazin-2-yl)-4-methylmorpholinium
chloride (DMT-MM) as a reagent (scheme 5). This enables the use of alcohols and water as solvents.
They are good solvents because they are very inexpensive and are much greener than the solvents
usually used in this kind of chemistry.
(Kunishima et al., 2001)
Scheme 5: Amide bond formation using DMT-MM
- 42 -
The same paper suggests that this method has higher selectivity for primary compared to secondary
amines and greater yields in comparison to simply using carbonyldiimidazole.
1.2.8 Ethyl chloroformate
Scheme 6: Production of mixed anhydride using ethyl chloroformate.
Ethyl chloroformate can be used in acid coupling reactions (scheme 6), which has the advantage of not
having to use the methyl ester, as opposed to the aminolysis reaction which does. Use of ethyl
chloroformate gives rise to the production of a mixed acid anhydride, which is a more reactive group
than the acid (McMurry, 2004). Heating is not required for the amide bond to be formed, thus
preventing the problems associated with aminolysis, such as degradation of materials, thus making it
easier to purify.
1.3.0 Co-germinants of Clostridium difficile spores:
As well as bile acids being germinants of C. difficile spores, there is a group of compounds, which
comprise of amino acids or analogues thereof, which are known as co-germinants. While it is
generally accepted that a suitable bile amide will germinate the C. difficile spore on its own, if a co-
germinant is added then this can help increase the germination rate of the spore; it is a synergistic
relationship between the two compounds. As a general rule, single co-germinants are not able to
induce germination without the presence of a bile acid analogue.
- 43 -
Table 2: List of amino acids and small molecular weight compounds which act as co-germinants or
inhibitors of C. difficile spore germination.
Glycine-compound 38
β–alanine-compound 39
γ –butyric acid-compound 40
Aminomethylphosphonic acid-
compound 41
An inhibitor of germination
L-alanine-compound 42
D-alanine-compound 43
L-cysteine-compound 44
- 44 -
L-phenylalanine-compound 45
L-serine-compound 46
Dodecylamine-compound 47
Glycine is probably the most well-known co-germinant of C. difficile but it would appear that there
can be a considerable amount of manipulation of the structure of the amino acid and it will still act as a
co-germinant. An important factor is simply how hydrophobic/hydrophilic the compound is. Examples
include β-alanine and γ-butyric acid (Table 2). One paper seemed to indicate that it is not the chain
length between the amino and carboxyl groups in the co-germinant which is important but the fact that
there is a carboxyl and a free primary amino group inherent in the amino acid (Howerton et al., 2010).
Another key point is that although it does not seem to matter how long the chain between the amino
group and the carboxylic acid is, if there is any branching in that chain then this dramatically reduced
the germination properties of the co-germinant. There are exceptions to this rule however, with L-
phenylalanine and L-arginine also being shown to be good co-germinants (Howerton et al., 2011).
One analogue of glycine, aminomethylphosphonic acid (Table 2), has been shown to actually inhibit
the germination of C. difficile. While it retains the overall negative charge, the replacement of the
carboxylic acid carbon with phosphorus is clearly the reason for this removal of activity. The same
literature suggest that if any modification is made to the free amino group of glycine, then this
abolishes any co-germinating ability, showing that the free amino group is as important, if not more
so, than having the free carboxylic acid moiety. Furthermore, if the glycine’s carboxylic acid is
- 45 -
methylated, this decreases germination ability even more (10 % of the original value), suggesting that
the free acid group is key to its co-germinating ability (Howerton et al., 2010).
L-Alanine (Table 2) is an amino acid which has been shown to be as good a co-germinant as glycine.
What is interesting is that, although the L form of alanine was a co-germinant, the D version was not a
co-germinant for C. difficile, therefore implying that the stereochemistry is very important in
determining whether the material is a co-germinant (Howerton et al., 2010). Therefore, it is probable
that stereochemistry is also important in germinating materials, much like the conversion of hydroxyls
from alpha to beta, as discussed earlier, negating any germinating ability.
L-Cysteine and L-phenylalanine are both as good at co-germinating C. difficile as glycine, yet L-serine
is inactive against the bacterium. The fact that L-phenylalanine is such a hydrophobic amino acid
implies that perhaps it will be attaching to a different receptor site to glycine, as it is difficult to
imagine both amino acids fitting in the same site. In other spore forming bacteria, where more is
known about the receptor sites, there are numerous different sites for differing reagents, so it is
plausible that C. difficile also has these different receptor sites (Howerton et al., 2010). All these
factors together seem to imply that there are many different factors for germination, and that only a
few of them need to be fulfilled before germination will take place.
While isolated amino acids will not affect germination on their own, if multiple groups of amino acids
are introduced into the bacterium’s environment (L-phenylalanine, L-arginine and glycine) then this
will promote germination of the bacteria. It has also been shown that the bacterium will germinate in
the presence of Ca-DPA (Calcium diplionic acid), a key chemical in controlling germination),
potassium ions and in the presence of the surfactant dodecylamine (table 2), with no bile acid
analogues present at all (Paredes-Sabja et al., 2008, Wilson, 1983).
1.4.1 Protecting groups for bile acids:
Due to the inherent nature and shape of bile acids, the C-3 hydroxyl group is much more open to
attack due to the steric hindrances which “protect” C-7 and C-12 to some extent.
- 46 -
Fig 14: Positions of the hydroxyls in compound 10.
There are quite a few reactive functional groups in the bile acid structure where reactions can take
place, so there is often the need for use of protecting groups, either the carboxylic acid groups or the
hydroxyl groups. For the hydroxyl groups the simplest way to protect them is through the use of
acetate groups, which are easy to prepare, highly stable and easily removed via hydrolysis when they
are no longer required (Kuhajda et al., 1996, Gao and Dias, 1999). As there is a CH2 (C4) unit in a 1,3
diaxial position, then this means that the 7-OH is more sterically hindered than the 12- OH, which is
itself more sterically hindered than the 3- OH (Davis and Joos, 2003). By varying the specific reaction
conditions, different groups can be protected/deprotected. Acetylation of the 3-OH, 7-OH and 12-OH
can be achieved by using Ac2O at room temperature. This protects all three of the OH groups. It is
relatively easy, however, to selectively remove the acetate group from the C-3 hydroxyl group. This
can be done through the use of K2CO3 in methanol at room temperature, with a yield of 99 % (Gao and
Dias, 1999). Therefore, if specific manipulation at 3-OH is required, there is no chance of production
of diprotected or triprotected compounds being formed as the other hydroxyl groups will still be
protected. Another method for acetylation is HCOOH/HClO4/55o/Ac2O (Malik et al., 1986). If
protection of both C-3 and C-7 is required, whilst leaving C-12 open for manipulation, this is
relatively easy to do as well.
Other possibilities are to convert the hydroxyl groups into ketones, which can be done chemically
using potassium chromate and sodium acetate. To convert specific hydroxyl groups to ketones using
bacteria, this can be achieved using Acinetobacter calcoaceticus (Giovannini et al., 2008).
10
- 47 -
To manipulate the hydroxyl groups, the carboxylic acid group will need to be protected to prevent any
unwanted side reaction from occurring. The easiest way to do this is to convert the acid into the
methyl ester. The conversion to the methyl ester can be done by several different methods; however,
the method of choice seems to be HCl/MeOH. Use of TMSCHN2 in methanol has been reported, with
the advantage of this method leaving already protected acetate hydroxyl groups unaffected (Gao and
Dias, 1999), as opposed to the HCl/MeOH which can cause cleavage of acid sensitive protecting
groups.
Another method for producing ketones is to use Jones’ reagent (CrO3) (Fieser and Rajagopalan, 1950)
and acetone. Once the ketone has been produced, it is possible to further protect it as a cyclic ketal by
the use of ethylene glycol. To produce the ketone specifically at C-7, the conditions needed are room
temperature and the use of AcCl and CH3OH. Conversion of alcohol to ketone at only the C-7 position
can also be achieved by use of NBA(N-bromoacetamide), acetone, H2O at room temperature for 3
hours (Gao and Dias, 1999).
There are also specific chemical protecting group reagents; these include tert-butyloxycarbonyl (Boc)
and 9-fluorenylmethyloxycarbonyl (Fmoc). One way of attaching a Boc group is to use di-tert-butyl
dicarbonate which is attacked by the nucleophilic amine to produce the protected amide.
Trifluoroacetates are another method of protecting hydroxyl groups in bile acids. It has been shown
however, that trifluoroacetates are much more prone to hydrolysis than acetate groups. The advantage
being however, that the terminal carboxylic acid does not need to be converted to the methyl ester first
before manipulation. Again, like the acetate protection groups, the solvent used dictates which
hydroxyl group is specifically protected (Gao and Dias, 1999). Reaction conditions often involve
TFAA, THF, then NaHCO3, MeOH and THF (Gao and Dias, 1999).
During protection with acetates, it was found that pure products could not be isolated without the use
of column chromatography, so a new methodology was required to get around this problem (Gao and
- 48 -
Dias, 1999). Use of formate as a protecting group meant that the product was in a crystalline state,
which was able to have characterised and well defined melting points.
1.5.1 Stereochemistry of bile acids.
Inversion of stereochemistry (fig 15) is very important in designing drugs and other molecules. One
form of a drug may be life-saving, whereas another isomer may have no effect/unwanted effect.
Conversion of hydroxyl groups from α to β can be done, specifically for the hydroxyl group at C-7
(Compound 48).
Fig 15: Inversion of stereochemistry at the 7 position in methyl cholate, compound 48.
This inversion of stereochemistry is achieved by first oxidising the alcohol to the ketone using
potassium dichromate and then performing a stereospecific reduction using potassium tert amyl
alcohol.
1.6.1 Polymer overview:
Synthetic polymers are prepared by the polymerisation of monomeric units. The easiest way to do this
is by a process called free radical polymerisation, which is the industry standard for about 40-45 % of
industrial polymers (Nesvadba, 2012). The advantages of free radical polymerisation compared with
any other form of polymerisation are that it is able to be performed in the majority of solvents and the
- 49 -
presence of trace oxygen (Nesvadba, 2012). There are many initiators of free radical polymerisation
but the most well-known (and used in this project) is AIBN (azo-isobutyronitrile) (Scheme 7). The
most important features of this azo class of initiators are the fact that they are symmetrical
dialkyldiazenes which have tertiary alkyl groups to stabilize the radical which is formed. By having
these features on the free radical initiator, the decomposition temperature of the compound can be
fine-tuned to the needs of the project. They also have a greater tendency (compared to other photo
initiators) to undergo chain transfer reactions. The propagating organic radicals are as a general rule,
planar. While it was stated earlier that free radical polymerisations are tolerant of trace levels of
oxygen, fully saturated oxygen environments do stop the polymerisation entirely, due to formation of
peroxy radicals being formed (Scheme 8).
Scheme 7: Mechanism of AIBN breakdown to produce a reactive radical species (Clayden, 2009).
Scheme 8; Peroxy radical formation by allowing free radical polymerisation to occur in an oxygen
environment.
- 50 -
1.6.2 Bile acid derived polymers.
The first literature relating to polymerisation of bile acids was in 1988 (not free radical) (Zuluaga et
al., 1999). This was carried out in toluene at 90-100 oC using p-toluene sulfonic acid as the catalyst to
induce ester formation. The initial bile acid was cholic acid and it was observed that some crosslinking
would occur at both positions 7 and 12; the amount of this crosslinking was directly related to the
temperature at which the material was heated. A lot of work has gone into the addition of
polymerizable groups onto either hydroxyl groups or the C24 carboxylic acid groups, and therefore
producing polymerizable bile acids. As stated earlier, these bile acid structures have a unique
amphilicity which means that they can potentially bind to the polymer matrix through both the
carboxylic acid side chain and the hydroxyl groups (Idziak et al., 1999).
Other research groups (Avoce et al., 2003) have worked on producing polymeric materials by
attaching amines with polymerizable units to the carboxylic acid tail (fig 16).
Fig 16: A polymerized bile acid (compound 49).
One possible way of producing a polymeric bile amide species is to attach an acryloyl group to the
hydroxyls located on the bile amide structure. The 3-OH is the most reactive of the three hydroxyl
groups and therefore would be preferentially attacked. If needed though, all three hydroxyl groups can
have acryloyl groups attached but the conditions required to do so are much harsher.
49
- 51 -
It is also possible to react elsewhere on the steroid compound. If ethylene glycol has been attached to
the carboxylic acid group to produce an ester it is then possible to react on the primary hydroxyl at the
end of the ethylene glycol chain (Zhu and Nichifor, 2002). If the carboxylic acid is not the required
attachment site, then this can be converted to the methyl ester and as well as protecting the acid group
from attack it will also improve the solubility of the material.
Polymethacrylates (fig 17) are widely used in biomedical technology; however, there is a problem
with their toxicity. When attached to bile acids and polymerised they produce super branched
macromolecules which are self-organising and possess good thermal and mechanical properties (Hao
et al., 2006). The main advantage of attaching a natural product to this methacrylate group is that the
biocompatibility of the overall compound is improved (Avoce et al., 2003).
Fig 17: Polymethacrylated cholic acid (compound 50)
1.6.3 Antimicrobial polymers
It is a well reported fact that the majority of bacterial cell walls are negatively charged. Therefore, if
the polymer is to be antimicrobial, the way to do this is to make the polymer itself positively charged;
thus the wide usage of polymers with quaternary ammonium ions incorporated into the structure
(Munoz-Bonilla and Fernandez-Garcia, 2012). There is a collection of polymers, known as protonated
50
- 52 -
polyethyleneimines which have a track record of killing bacteria because of their unique structures
(Dhende et al., 2011) fig 18.
Fig 18; Polyethyleneimines, an antimicrobial cationic polymer (compound 51).
It was found that when polymers have a charge to chain length ratio which is roughly equal (carbons
between charged nitrogen’s), this improved its antimicrobial function. However, it also increased its
toxicity towards mammalian cells (fig 18).
Polymers are ideal as antimicrobial agents due to their low toxicity, plus the fact they are reusable as
no materials are released and it is inherently antimicrobial. Due to the mode of action of these
materials (due to quaternized nitrogen), the risk of bacterial resistance is minimal because the cell is
completely destroyed, thus minimal chance of survival. In an age when the treatments are becoming
less and less effective, prevention is better than cure. The use of a antimicrobial surface would reduce
the microbial load on said surface and would be integral to a hygiene routine which would ultimately
break the nosocomial infection loop (Page et al., 2009). However, eventually it could be surmised that
resistance to quaternized material would eventually occur and this could present another problem in
the future. This could potentially be minimised by use of several different alkylating agents for
quaternization so resistance is slowed down considerably.
PDMAEMA (poly N, N-dimethylaminoethyl methacrylate) (fig 19) has been shown to prevent any
effect of the cytotoxins of C. difficile once they have been released (Munoz-Bonilla and Fernandez-
Garcia, 2012). Although this is an interesting finding, the fact that this cannot be taken orally means
that it is not the optimum solution to the problem currently faced.
51
- 53 -
Fig 19: PDMAEMA (poly N, N-dimethylaminoethyl methacrylate, compound 52), an antimicrobial
polymer (Lu et al., 2007).
1.7.1 Benzophenone introduction:
Ultraviolet light has been the primary methodology for the curing of coatings since the early 1980s.
Benzophenone has been widely used as the type 2 initiator (hydrogen abstraction-type) of such curing
reactions (Cheng and Shi, 2011).
An alternative way of producing polymeric materials is through the use of UV radiation to produce
hydrogen abstraction off a triplet benzophenone, thus producing free radicals which can come together
to form a polymer (Lin et al., 1988). The photochemistry of benzophenone has been heavily
investigated because of its ability to be both an electron acceptor, hydrogen abstracting agent or to
work as an energy transfer donor (Muldoon et al., 2001).
One of the advantages of this hydrogen abstraction is the fact that although oxygen does have a minor
inhibitory effect on the reaction, it is not completely inhibitory like in most other polymerisation
techniques (excluding AIBN methodology discussed earlier). The choice of solvents is key in this
reaction as it defines the molecular interactions which will occur (Castro et al., 2000).
52
- 54 -
Benzophenones have an interesting chemistry. Both the phenyl groups interact with the carbonyl
group through both σ and π bonds, creating a molecular orbital which incorporates the entire structure.
This produces a carbonyl group which loses its individual character because of the delocalization, thus
allowing hydrogen abstraction to occur more easily (Castro et al., 2000). One of the most developed
reactions involves the use of isopropanol as the solvent, the reaction scheme for the ground state and
electronically excited radical are shown in scheme 9.
- 55 -
Scheme 9; Ground and excited state of benzophenone, with all possible outcomes upon exposure to
UV light. R = Phenol.
- 56 -
Benzophenone absorbs wavelengths which are approximately 350 nm. This promotes one electron
from a non-bonding sp2 orbital on the oxygen to and anti-bonding π
* orbital on the carbonyl group.
This causes the oxygen to become electron deficient and to become electrophilic, interacting with C-H
σ bonds, thus abstracting the hydrogen. If any group comes near the carbonyl in this excited state,
electron transfer can occur and the ketyl radicals formed can recombine to produce a new C-C bond
(Dorman and Prestwich, 1994).
If benzophenone is left in direct sunlight in solution (ethanol), this is enough for a reaction to occur.
The major products of this sort of reaction are benzopinacol and isobenzopinacol (fig 20) (Pitts et al.,
1959). However, once again, the choice of solvents is highly important with cyclohexene completely
retarding the benzopinacol formation. It was further found that cross coupling reactions would only
occur if hydrogen abstraction was easily achieved, most noticeably this occurs with alcohols, toluene
and cumene (Weiner, 1971). It has also been shown (Qu, 2002), that it is easier for the benzophenone
to abstract from tertiary bonded hydrogen, than a secondary bonded hydrogen, which in turn is easier
to be abstracted than a primary (Dorman and Prestwich, 1994).
Fig 20: Structure of benzopinacol (compound 53) and isobenzopinacol (compound 54), two products
from photo initiation of benzophenone.
To produce a material with germinating ability and be photochemically active, amino-benzophenone
can be chemically attached to a bile acid. Thus a potential germinant material would be attached to a
material (benzophenone) which could be attached to any organic surface by subjecting the material to
ultraviolet light. This would be another way of producing a polymeric potential
germinant/antimicrobial surface which could be applied almost anywhere required.
53 54
- 57 -
As an added advantage to the already proven properties of benzophenone as a photo initiator, literature
suggests that poly (benzophenone) can be potentially antimicrobial against S. aureus and E. coli,
which could mean that any potential germinant polymer may also end up killing other unwanted
bacteria as well (Hong and Sun, 2009). Also in that same literature it was suggested that incorporation
of the benzophenone moiety into the polymer structure can cause mechanical weakness in the
polymer’s properties.
1.8.1 Quaternizable materials/antimicrobials:
A lot of work has been done on the introduction of a polycationic chain into a steroid structure
scaffold with the hope of it being antimicrobial (Aher et al., 2009, Tiller et al., 2001). Active
compounds are able to disrupt and/or make the cell membranes of prokaryotes much more permeable.
There has been a lot of research going into making dry state polycations. If dried, polycations are
rendered useless, as they cannot penetrate the cell membrane and disrupt it. If there was a long spacer
group however, the polycation would be able to reach the membrane and therefore cause the damage,
with biological activity only being present if there is also a polar head group (Bouloussa et al., 2008,
Aher et al., 2009). It has been reported that the biological activity of quaternary ammonium
compounds is very much dependent on the organic species which is attached the nitrogen, the number
of nitrogens present within the molecule and the counter ion to the positively charged nitrogen
(Munoz-Bonilla and Fernandez-Garcia, 2012).
The mechanism of action of cationic materials has been studied and the process seems to go thus:
adsorption onto cell surface, diffusion through the cell wall, binding to cytoplasmic membrane, release
of K+ ions and other cytoplasmic constituents which leads to precipitation of cells contents due to loss
of ionic integrity (Dhende et al., 2011) and thus death (Ikeda et al., 1984). This absorption will occur
quicker with polymers as opposed to monomer units because of the higher density of cationic charges
in a single area.
- 58 -
It has been shown that polymers which contain quaternary ammonium moieties can potentially be
antimicrobial. This is a potentially important area (Perichaud et al., 2001). However, research also
shows that if this polymer is cross-linked or is insoluble, then any antimicrobial activity is lost as the
material has not got the range of movement to find the bacterial cell wall and disrupt it (Tiller et al.,
2001). This method of making a surface antibacterial has the distinct advantage that there is no chance
of the bacteria developing a resistance to it as to do so would require alterations to the bulk membrane
structure which is a very difficult task for a cell to undertake (Guan et al., 2000). Therefore, in a
polymer surface, this would be ideal as any microorganisms that landed on the surface would be
immediately eradicated. It has the other advantage of not being selective, so as well as being active
against C. difficile it would kill other unwanted harmful microorganisms. In fact, there have been
polymers produced (fig 21) which are aimed at S. aureus which have been alkylated and have been
successful at killing the bacteria.
Fig 21: An antimicrobial polymer, with quaternary nitrogen ions, used in the prevention of S. aureus.
Modified from (Tiller et al., 2001) (compound 55).
Another material which has been used as an antimicrobial agent is copper. Although clearly not a
quaternized material it has been shown (Wheeldon et al., 2008b) that copper surfaces are antimicrobial
within a certain time frame (24-48 hours). What is interesting is that although usually the
antimicrobials were aimed at killing the vegetative cells after germination, copper has been shown to
also be sporicidal.
55
- 59 -
The problem with current literature is that, although work has been done on antimicrobial agents and
other scientists have done work on germinatory effects of bile acids on C. difficile, none that could be
found that specifically worked on both germination and then killing of C. difficile cells/spores. The
antimicrobials which have been made (not bile acid based) are always tested on other gram positive
bacteria, so there is clearly a gap in the literature which this project hopes to fill.
1.8.2 Antimicrobial bile amides.
The advantages of polyamines (in particular) are found to be generally of low toxicity and have a
defined structure for pharmaceutical characterization (Aher et al., 2009). An example of an amino
sterol with antimicrobial properties is shown below in the form of squalamine, a natural bile acid
originally found in the stomach tissue of the dogfish shark (Randazzo et al., 2009) (fig 22). There are
several mimics of squalamine which have been reported (Kikuchi et al., 1997), all based on steroidal
structures, including cholic, deoxycholic and lithocholic acid. Although the exact mechanism of
antibacterial activity in squalamine is unknown, it is postulated that it works by perforating the
membranes of the bacteria.
Hydrophobicity of the bile acid compound is important in antimicrobial action, with there being an
optimum logP value. Basicity of the bile acid analogues proved to be important for antimicrobial
activity and, as with hydrophobicity, there is an optimum value. As well as activity against gram
positive bacteria, there has been a large amount of work done on the antimicrobial activity of bile acid
analogues against gram negative bacteria. This same research (Cravotto et al., 2005), has shown that
some dimeric bile acids are antifungal and anti-proliferative. Gram-positive bacteria differ from gram-
negative bacteria in the structure of their cell walls. The cell walls of gram-positive bacteria are made
up of twenty times as much murein or peptidoglycan than gram-negative bacteria. These complex
polymers of sugars and amino acids cross-link and layer the cell wall. The outer cell membrane of a
prokaryote has an overall negative charge (Guan et al., 2000).
- 60 -
Fig 22: Squalamine (compound 56), a steroid based antibiotic, effective against gram negative rods
and gram positive cocci (Aher et al., 2009).
Another material which has shown antimicrobial activity, specifically against gram positive bacteria is
lithocholyl-N-2-(aminoethyl) amide Fig 23.
Fig 23: Lithocholyl-N-2-(2-aminoethyl) amide (compound 57), a bile amide which has shown in vitro
activity against gram positive bacteria (Ahonen et al., 2010).
The aim of this project is, at the very least, to better understand the important structural characteristics
which are essential for germination of C. difficile. By building up a structural database using both
lithocholic, deoxycholic and to a lesser extent, cholic acid, the important factors should emerge. If a
compound which is a good germinant can be found, then incorporating this compound into a
56
57
- 61 -
polymeric surface (while still retaining germinating ability), this would be key. There is also a need to
be able to kill the vegetative cells, so incorporation of an antimicrobial entity into this polymer would
be required.
- 62 -
2.0 Experimental:
2.1.1 Instrumentation
Proton NMR were obtained using a Bruker AC250 instrument (250 MHz) as solutions and referenced
from δ TMS = 0.00 pm. Carbon NMR (CPD and DEPT) were obtained using a Bruker AC250
instrument (62.9 MHz) as solutions and referenced from δ TMS = 0.00 pm. All analysis of carbon
NMR incorporates both CPD and DEPT data and used data from (Waterhous et al., 1985) for
assignments of the carbon NMR. Infrared spectra were recorded as KBr discs on a Mattson 3000 FTIR
spectrometer or Thermo Scientific Nicolet 1s5 with 1D5 ATR attachment as a solid sample.
Atmospheric pressure chemical ionisation mass spectrometry (APCI) was obtained using a Hewlett-
Packard 5989B quadropole instrument connected to an electrospray 59987A unit with an APCI
accessory and automatic injection using a Hewlett-Packard 1100 series auto sampler. ESI spectra were
obtained using a Thermofisher LTQ orbitrap XL at the EPSRC facility at Swansea University. Melting
points were obtained using a Reichert-Jung Thermo Galen hot stage microscope and are corrected.
The work done on the anthranilamide based materials was done using a Leica Microsystems GmbH
TCS SP5 II system with an upright DMI6000B microscope. The UV source for the benzophenone
attachment work was a Proxima Direct 36 Watt UV, 220-240 V/50 Hz Nail curer. All work was
carried out at Aston University, either in the medicinal chemistry laboratory or the NMR suite. All
experiments were COSHH assessed, using the standard practices in the medicinal chemistry
laboratory. All materials were sourced from Sigma-Aldrich or Fisher with no further purification. For
the ethyl chloroformate amide formation reactions, all materials analysed by TLC (thin layer
chromatography) had an Rf of 0.2-0.3 and were all single spots in an eluent of 8:2 EtOAc/MeOH. For
quaternization reaction materials analysed by TLC 100 % MeOH, single spots with Rf of 0.1-0.2.
When removing solvents under vacuum on the rotary evaporator, ethyl acetate was taken off at 55 oC.
All TLCs were visualised using vanillin as a stain. All synthetic chemistry work was done by Matthew
Justin Hird. All biological testing work was done between Christian Lowden, Kristian Poole and
Amber Lavender under the supervision of Dr Tony Worthington.
- 63 -
When referring to carbon numbers in the experimental, the numbers are associated with the labelled
bile acid below (fig 24)
Fig 24: The carbon numbering system for a steroid based structure (in this case cholic acid). This
numbering system will be used in the bile acid derived compounds reported in this thesis.
Due to the nature of the structures associated with this project, full assignment can be very difficult. In
particular the majority of the steroid based hydrogens will not be explicitly stated, only the key peaks
which define compound structure. There are a large amount of overlapping peaks starting from ~ 0.95
ppm and finishing at ~ 2.50 ppm which are associated with protons attached to carbons 1, 2, 4-6, 8-11,
14-17, and 20-23. For ease, as these peaks are not affected by structural changes they will not be
explicitly dealt with and a range will be provided.
- 64 -
2.2.1 Ester preparation
Preparation of methyl lithocholate (Comp 58)
Lithocholic acid (5.0 g, 0.01329 mol) was added to methanol (90 mL) to produce a suspension. Acetyl
chloride (0.5 mL, 0.006 mol) was then added. The solution was heated and stirred at 80 oC for 40
minutes as a homogeneous solution, then allowed to cool overnight in an ice bath. This was added to
water (150 mL) and the resulting precipitate was collected by filtration, washed with water (3 x 20
mL) and dried under vacuum. TLC EtOAc: MeOH 4:1 Rf 0.25 (single spot).
Yield; 5 g, 0.01328 mol, 96.5 %.
Melting point: 75-76 oC. Lit 71-73
oC, (Huong et al., 2009).
1H NMR (CDCl3) (250 MHz) δ= 0.66 (s, 3H, 18-CH3), 0.94 (s, 3H, 19-CH3), 0.95-2.39 (m, 33H,
steroidal backbone CH/CH2), 3.65 (m, 1H, 3-CH), 3.69 (s, 3H, O-CH3) ppm.
13C NMR (CDCl3) (62.9 MHz) δ= 12.0 (CH3, C18), 18.6 (CH3, C21), 20.8 (CH2, C11), 23.7 (CH3,
C19), 24.2 (CH2, C15), 26.4 (CH2, C7), 27.2 (CH2, C6), 28.2 (CH2, C16), 30.5 (CH2, C2), 30.9 (CH2,
C22), 31.0 (CH2, C23), 34.5 (C, C10), 35.3 (CH, C20), 35.4 (CH2, C1), 35.8 (CH, C8), 36.4 (CH2,
C4), 40.1 (CH2, C12), 40.4 (CH, C9), 42.0 (CH, C5), 42.7 (C, C13), 51.5 (O-CH3), 55.9 (CH, C17),
56.5 (CH, C14), 71.9 (CH, C3), 174.8 (CO, C24) ppm.
- 65 -
IR; 3347 (OH stretch), 2937 (C-H), 2859 (O-CH3), 1733 (C=O), 1640, 1436, 1206, 1043 (R2CH-OH)
cm-1
.
Preparation of methyl deoxycholate (comp 59)
Deoxycholic acid (5.0 g 0.01 mol) was dissolved in methanol (30 mL) and treated with acetyl chloride
(0.5 mL, 0.006 mol). The solution was heated and stirred at 80 oC for 40 minutes then allowed to cool
overnight in an ice bath. The resultant crystals were collected by vacuum filtration to produce a white
crystalline powder. This was washed with water (2 x 20 mL) and dried under vacuum. TLC EtOAc:
MeOH 4:1 Rf 0.25 (single spot).
Yield; 1.81 g, 0.004 mol, 35 %.
Melting point: 70-72 oC. Lit 67-70
oC (Anelli et al., 2009).
1H NMR (CDCl3) (250 MHz) δ= 0.68 (s, 3H, 18-CH3), 0.91 (s, 3H, 19-CH3), 1.00-2.38 (m, 33H,
steroidal backbone CH/CH2), 3.60 (m, 1H, 3-CH), 3.67 (s, 3H, O-CH3), 3.99 (s, 1H, 12-CH) ppm.
13C NMR (CDCl3) (62.9 MHz) δ= 12.8 (CH3, C18), 17.3 (CH3, C21), 23.2 (CH3, C19), 26.6 (CH2,
C15), 26.1 (CH2, C7), 27.1 (CH2, C6), 27.4 (CH2, C16), 28.7 (CH2, C11), 30.5 (CH2, C2), 30.9 (CH2,
C23), 31.1 (CH2, C22), 33.7 (CH, C9), 34.1 (C, C10), 35.1 (CH2, C1), 35.2 (CH, C20), 36.0 (CH2,
C4), 36.4 (CH, C8), 42.0 (CH, C5), 46.5 (C, C13), 47.3 (CH, C17), 48.3 (CH, C14), 51.5 (O-CH3),
71.8 (CH, C3), 73.1 (CH2, C12), 174.7 (CO, C24) ppm.
- 66 -
IR; 3474 (OH stretch) 2985 (C-H), 2852 (O-CH3), 1743 (C=O), 1450, 1380, 1040 (R2CH-OH) cm-1
.
Preparation of 2-hydroxyethyl (4R)-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-
2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoate
(compound 60)
Lithocholic acid (0.5 g, 0.001 mol) was dissolved in ethylene glycol (15 mL 0.24 mol) and acetyl
chloride (0.1 mL 0.001 mol) was added. The reaction was sealed under argon and heated to 100 oC
overnight. Water (50 mL) was added and the solution was allowed to cool for 1 hour in an ice bath,
which produced precipitate which was washed with water (3 x 20 mL) and dried under vacuum. TLC
EtOAc: MeOH 4:1 Rf 0.25 (single spot).
Yield; 0.43 g, 0.001 mol, 78 %.
Melting point: 75-77 oC.
1H NMR (CDCl3) (250 MHz) δ= 0.63 (s, 3H, 18-CH3), 0.91 (s, 3H, 19-CH3), 0.95-2.47 (m, 33H,
steroidal backbone CH/CH2), 3.56 (m, 1H, 3-CH), 3.83 (t, 2H, CH2, J= 5.0), 4.21 (t, 2H, CH2, J= 5.0)
ppm.
13C NMR (CDCl3) (62.9 MHz) δ= 12.0 (CH3, C18), 18.2 (CH3, C21), 20.8 (CH2, C11), 23.3 (CH3,
C19), 24.2 (CH2, C15), 26.4 (CH2, C7), 27.2 (CH2, C6), 28.2 (CH2, C16), 30.5 (CH2, C2), 30.9 (CH2,
C22), 31.1 (CH2, C23), 34.5 (C, C10), 35.3 (CH, C20), 35.8 (CH2, C1), 36.4 (CH, C8), 40.1 (CH2,
C4), 40.4 (CH2, C12), 41.0 (CH, C9), 42.0 (CH, C5), 42.7 (C, C13), 55.9 (CH, C17), 56.5 (CH, C14),
- 67 -
61.3 (CH2, C(=O)OCH2 or CH2OH), 65.97 (CH2, C(=O)OCH2 or CH2OH), 71.9 (CH, C3), 174.7 (CO,
C24) ppm.
IR= 3334 (OH), 2857 (alkyl), 1727.11 (C=O), 1441, 1168 (C-O ester stretch), 1000 (R2CH-OH) cm-1
.
MS (+ESI) m/z= Found 438.3579 (M+NH4)+; calculated for C26H48NO5 438.3578; 0.3 ppm.
Preparation of 2-hydroxyethyl (4R)-4-[(3R,10S,12S,13R,17R)-3,12-dihydroxy-10,13-dimethyl-
2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoate
(compound 61).
Deoxycholic acid (0.5 g, 0.001 mol) was dissolved in ethylene glycol (5 mL 0.06 mol) and acetyl
chloride (0.1 mL 0.001 mol) was added. The reaction was sealed under argon and heated to 100 oC
overnight. Water (50 mL) was added and the solution was allowed to cool for 1 hour in an ice bath,
which produced a precipitate which was washed with water (3 x 20 mL) and dried under vacuum. TLC
EtOAc: MeOH 4:1 Rf 0.25 (single spot).
Yield; 0.21 g, 0.0004 mol, 38 %.
Melting point: 128-131 oC.
1H NMR (CDCl3) (250 MHz) CDCl3 δ= 0.69 (s, 3H, 18-CH3), 0.92 (s, 3H, 19-CH3), 0.98 (d, 3H, 21-
CH3, J = 5.0), 1.00-2.43 (m, 33H, steroidal backbone CH/CH2), 3.39 (broad s, 1H, 3-CH), 3.65 (m, 1H,
12-CH), 3.85 (t, 2H, CH2, J= 5.0), 4.20 (t, 2H, CH2, J= 5.0) ppm.
- 68 -
13C NMR (CDCl3) (62.9 MHz) δ= 12.0 (CH3, C18), 18.2 (CH3, C21), 20.8 (CH2), 23.3 (CH3, C19),
24.2 (CH2, C15), 26.4 (CH2, C7), 27.1 (CH2, C6), 28.2 (CH2, C16), 30.5 (CH2, C11), 30.9 (CH2, C2),
31.1 (CH2, C23), 34.5 (CH, C9), 35.3 (C, C10), 35.8 (CH2, C1), 36.4 (CH, C20), 40.1 (CH), 40.4 (C,
C13), 42.0 (CH2 C5), 42.7 (CH), 55.9 (CH2, C(=O)OCH2 or CH2OH), 56.5 (CH2, C(=O)OCH2 or
CH2OH), 61.3 (CH2), 65.9 (CH, C3), 71.8 (CH2, C12), 174.7 (CO, C24) ppm.
IR= 3509 (OH), 3309 (OH), 2925 (alkyl), 2857 (alkyl), 1718 (C=O), 1449, 1356, 1292, 1189 (C=O
ester stretch), 1027 (R2CH-OH) cm-1
.
MS (+ESI) m/z= Found 454.3527 (M+H)
+; calculated for C26H48NO5 454.3527; 0.0 ppm.
2.3.1 Amide formation
Preparation of (4R)-N-(2-dimethylaminoethyl)-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-
2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-
yl]pentanamide (compound 62)
A mixture of lithocholic acid (1.0 g 0.005 mol) and N, N-dimethylethylenediamine (0.16 mL 0.001
mol) was dissolved in toluene (20 mL). The solution was heated at reflux for 24 hours. Analysis by
TLC indicated that the reaction had not gone to completion so a further (0.32 mL 0.003 mol) of N, N-
dimethylethylenediamine was added. Water (100 mL) was added to the solution, causing material to
precipitate out. The precipitate was collected by vacuum filtration and the crude product was
recrystallized from ethyl acetate to produce a white powder.
- 69 -
Yield 0.85 g, 0.001 mol, 69%.
Melting point: Not recorded.
1H NMR (250 MHz) (CDCl3) δ= 0.64 (s, 3H, 18-CH3), 0.92 (s, 3H, 19-CH3), 0.94-2.30 (m, 33H,
steroidal backbone CH/CH2), 2.21 (s, 6H, 2 x CH3), 2.42 (t, 2H CH2 J=5.0), 3.34 (q, 2H, CH2, J= 7.5),
3.63 (m, 1H, 3-CH), 6.20 (broad s, 1H, NH) ppm.
13C NMR (62.9 MHz) (CDCl3) δ= 12.0 (CH3, C18), 18.3 (CH3, C21), 20.8 (CH2, C11), 23.3 (CH3,
C19), 24.2 (CH2, C15), 26.4 (CH2, C7), 27.2 (CH2, C6), 28.2 (CH2, C16), 30.5 (CH2, C2), 31.7 (CH2,
C22), 33.5 (CH2, C23), 34.5 (C, C10), 35.3 (CH, C20), 35.5 (CH2, C1), 35.8 (CH, C8), 36.4 (CH2,
C4), 36.6 (CH2, NHCH2 or CH2N(CH3)2), 40.2 (CH2, C12), 40.4 (CH, C9), 42.1 (CH, C5), 42.7 (C,
C13), 45.1 (CH3, NCH3), 56.0 (CH, C17), 56.5 (CH, C14), 57.9 (CH2, NHCH2 or CH2N(CH3)2), 71.8
(CH, C3), 173.7 (CO, C24) ppm.
IR; 3377 (OH), 3293 (NH) 2929 (C-H), 2870 (C-H), 1646 (C=O), 1543, 1445 cm-1
.
MS (+ESI) m/z= Found 447.3943 (M+H)+; calculated for C28H51N2O2 447.3945; 0.5 ppm.
- 70 -
Preparation of (4R)-4-[(3R,10S,12S,13R,17R)-3,12-dihydroxy-10,13-dimethyl-
2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]-N-(2-
dimethylaminoethyl)pentanamide (compound 63)
A mixture of deoxycholic acid (1.0 g 0.002 mol) and N, N-dimethylethylenediamine (0.16 mL 0.001
mol) was dissolved in toluene (20 mL). The solution was heated at reflux for 24 hours. Analysis by
TLC (EtOAc: MeOH 4:1) indicated that the reaction had not gone to completion so a further (0.16 mL
0.001 mol) of N, N-dimethylethylenediamine was added and left for a further 24 hours. Solvent was
then evaporated under reduced pressure, then triturated with hot water (~10 mL) and washed with
water (3 x 20 mL), then dried under vacuum. TLC EtOAc: MeOH 4:1 Rf 0.25 (single spot).
Yield 0.310 g, 0.0006 mol, 24%.
Melting point: Not recorded.
1H NMR (CDCl3) (250 MHz) δ= 0.68 (s, 3H, 18-CH3), 0.91 (s, 3H, 19-CH3), 1.01 (d, 3H, 21-CH3, J=
6.5), 0.97-2.43 (m, 33H, steroidal backbone CH/CH2), 2.26 (s, 6H, 2 x CH3), 2.46 (t, 2H, CH2, J=
5.0), 3.34 (m, 2H, CH2, J=5.0), 3.61 (m, 1H, 3-CH), 3.97 (broad singlet, 1H, 12-CH), 6.44 (broad
singlet, 1H, NH) ppm.
13C NMR (CDCl3) (62.9 MHz) δ= 12.7 (CH3, C18), 17.5 (CH3, C21), 23.1 (CH3, C19), 23.6 (CH2,
C15), 26.1 (CH2, C7), 27.1 (CH2, C6), 27.8 (CH2, C16), 28.5 (CH2, C11), 30.5 (CH2, C2), 31.6 (CH2,
C23), 33.3 (CH2, C22), 33.6 (CH, C9), 34.1 (C, C10), 35.2 (CH2, C1), 36.0 (CH, C20), 36.4 (CH2,
- 71 -
C4), 42.0 (CH, C5), 44.9 (C, C13), 46.5 (CH3, N-CH3), 47.3 (CH, C17), 48.2 (CH, C14), 57.9 (CH2
NHCH2 or CH2N(CH3)2), 71.7 (CH, C3), 73.0 (CH2, C12), 172.7 (CO, C24) ppm.
IR= 3305, (OH), 2929 (alkyl), 2861 (alkyl), 1720 (C=O), 1044 (R2CH-OH) cm-1
MS (+ESI) m/z= Found 463.3888 (M+H)+; calculated for C28H51N2O3 463.3894; 1.3 ppm.
Preparation of (4R)-N-[3-(dimethylamino)propyl]-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-
dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-
yl]pentanamide (compound 64)
Following the method of (Liu et al., 2001). A mixture of methyl lithocholate (0.5 g 0.001 mol) and 3-
dimethylamino-propylamine (3 mL, 0.02 mol) was heated and stirred at 140 °C for 24 hours in an
argon environment. Analysis by TLC (thin layer chromatography, 8:2 ethyl acetate/methanol)
indicated that the starting material had been consumed. Ice water (3 mL) was added to the material and
left to stir for two hours at room temperature. The resulting solid was then collected by filtration,
washed with water (3 x 20 mL) and left to dry overnight under vacuum to produce off-brown crystals.
TLC EtOAc: MeOH 4:1 Rf 0.25 (single spot).
Yield, 0.41 g, 0.0008 mol, 69 %.
Melting point: 177.0-178.3°C.
- 72 -
1H NMR (CDCl3) (250 MHz) δ= 0.64 (s, 3H, 18-CH3), 0.92 (d, 3H, 19-CH3), 0.95-2.20 (m, 33H,
steroidal backbone CH/CH2), 2.25 (s, 6H, 2 x CH3), 2.39 (t, 2H, CH2, J=6.3), 3.33 (q, 2H, CH2, J=5.6),
3.63 (m, 1H, 3-CH), 6.95 (s, 1H, NH) ppm.
13C NMR (CDCl3) (62.9 MHz) δ= 12.0 (CH3, C18), 18.3 (CH3, C21), 20.8 (CH2, C11), 23.3 (CH3,
C19), 24.2 (CH2, C15), 24.3 (CH2, C7), 26.4 (CH2, side chain CH2CH2CH2), 27.1 (CH2, C6), 28.2
(CH2, C16), 30.5 (CH2, C2), 31.7 (CH2, C22), 34.5 (C, C10), 35.3 (CH, C20), 35.5 (CH2, C1), 35.8
(CH, C8), 36.4 (CH2, C4), 40.1 (CH2, C12), 40.3 (CH, C9), 42.0 (CH, C5), 42.7 (C, C13), 43.0 (CH3
N-CH3), 55.3 (CH, C17), 55.9 (CH, C14), 56.4 (CH2 NHCH2 or CH2N(CH3)2), 71.9 (CH, C3), 174.9
(CO, C24) ppm.
IR= 3310-3318 (OH-NH), 2730 (alkyl), 2859 (alkyl), 2946 (alkyl), 1648 (C=O), 1047 (CH-OH) cm-1
.
MS (ES +APCI) m/z= Found 461.4105 (M+H)+; calculated for C29H53N2O2 461.4107; 0.4 ppm.
Preparation of (4R)-4-[(3R,10S,12S,13R,17R)-3,12-dihydroxy-10,13-dimethyl-
2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]-N-[3-
(dimethylamino)propyl]pentanamide (compound 65)
A mixture of methyl deoxycholate (0.5 g 0.002 mol) and 3-dimethylamino-1-propylamine (3.63 mL
0.03 mol) was heated at 100 oC for 5 days then allowed to cool to ambient temperature. Ice water (40
mL) was then added and left to stir for 2 hours and the precipitate collected by vacuum filtration,
- 73 -
washed with water (3 x 20 mL) and dried under vacuum. TLC EtOAc: MeOH 4:1 Rf 0.25 (single
spot).
Yield, 0.41 g, 0.0008 mol, 73 %.
Melting point: 123-127 oC.
1H NMR (CDCl3) (250 MHz) δ= 0.64 (s, 3H, 18-CH3), 0.92 (s, 3H, 19-CH3), 1.00-2.23 (m, 33H,
steroidal backbone CH/CH2), 2.23 (s, 6H, 2 x CH3), 2.42 (t, 2H, CH2, J= 5.0), 3.325 (q, 2H, CH2,
J=7.5), 3.63 (m, 1H, 3-CH), 6.21 (broad singlet, 1H, NH) ppm.
13C NMR (CDCl3) (62.9 MHz) δ= 12.0 (CH3, C18), 18.3 (CH3, C21), 20.8 (CH2, C11), 23.3 (CH3,
C19), 24.2 (CH2, C15), 26.4 (CH2, C7), 27.2 (CH2, C6), 28.2 (CH2, C16), 30.5 (CH2, C2), 31.7 (CH2,
C22), 33.5 (CH2, C23), 34.5 (C, C10), 35.3 (CH, C20), 35.5 (CH2, C1), 35.8 (CH, C8), 36.4 (CH2,
C4), 36.6 (CH2), 40.2 (CH2, C12), 40.4 (CH, C9), 42.1 (CH, C5), 42.7 (C, C13), 45.1 (CH2, NHCH2 or
CH2N(CH3)2), 56.0 (CH, C17), 56.5 (CH2, NHCH2 or CH2N(CH3)2), 57.9 (CH, C14), 71.8 (CH, C3),
173.7 (CO, C24) ppm.
IR= 3376 (NH and OH), 2938 (alkyl), 2861 (alkyl), 1643 (C=O), 1544, 1444, 1378, 1045 (R2CH-OH)
cm-1
.
MS (+ES APCI) m/z= Found 477.4064 (M+H)+; calculated for C29H53N2O3 477.4056; 1.7 ppm.
- 74 -
Preparation of 1-(4-butylpiperazin-1-yl)-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-
2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentan-1-
one (compound 66)
Following a modified method (Fini et al., 1992), lithocholic acid (0.5 g 0.001 mol) was dissolved in
tetrahydrofuran (20 mL) with triethylamine (0.4 mL 0.002 mol). The solution was cooled for 10
minutes in cold water. Ethyl chloroformate (0.2 mL 0.001 mol) was then added dropwise over a ten
minute period. Once added, the cold water was removed and the solution was stirred for 2 hours. After
2 hours, solvent removed under reduced pressure then re-dissolved in dichloromethane and washed
with water. 1-Butylpiperazine (0.25 mL 0.001 mol) was added and the solution was stirred for 24
hours. Water (50 mL) was then added and the solution extracted with ethyl acetate (3 x 50 mL). The
organic layers were combined and washed with saturated sodium hydrogen carbonate solution (3 x 50
mL). The organic layer was dried over magnesium sulfate. Solvent was evaporated under reduced
pressure. TLC EtOAc: MeOH 4:1 Rf 0.2 (single spot).
Yield; 0.16 g, 0.0003 mol, 23.5 %.
Melting point: 172.7-173.1 oC.
1H NMR (CDCl3) (250 MHz) δ= 0.61 (s, 3H, 18-CH3), 0.88 (s, 3H, 19-CH3), 1.00-2.25 (m, 33H,
steroidal backbone CH/CH2), 1.34 (m, 4H, CH2), 2.35 (m, 6H, CH2), 3.45 (broad singlet, 2H, CH2),
3.60 (broad singlet, 2H, CH2) ppm.
- 75 -
13C NMR (CDCl3) (62.9 MHz) δ= 12.0 (CH3, C18), 14.0 (CH3, CH2CH3), 18.5 (CH3, C21), 20.6 (CH2,
C11), 20.8 (CH2), 23.3 (CH3, C19), 24.2 (CH2, C15), 26.4 (CH2, C7), 27.2 (CH2, C6), 28.2 (CH2,
C16), 28.8 (CH2), 30.2 (CH2, C2), 30.5 (CH2, C22), 31.4 (CH2, C23), 34.5 (C, C10), 35.3 (CH, C20),
35.6 (CH2, C1), 35.8 (CH, C8), 36.4 (CH2, C4), 40.1 (CH2, C12), 40.4 (CH, C9), 41.4 (CH, C5), 42.1
(C, C13), 42.8 (CH2), 45.6 (CH2), 52.9 (CH2), 53.4 (CH2), 53.4 (CH2), 56.0 (CH, C17), 56.5 (CH,
C14), 58.3 (CH2), 71.7 (CH, C3), 172.1 (CO, C24) ppm.
IR= 3381, (OH), 2916 (alkyl), 2852 (alkyl), 1616 (C=O), 1437, 1253, 1036 (R2CH-OH) cm-1
.
MS (+ESI) m/z= Found 501.4410 (M+H)+; calculated for C32H57N2O2 501.4415; 0.9 ppm.
Preparation of (4R)-1-(4-butylpiperazin-1-yl)-4-[(3R,10S,12S,13R,17R)-3,12-dihydroxy-10,13-
dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-
yl]pentan-1-one (compound 67)
Deoxycholic acid (0.5 g 0.001 mol) was dissolved in tetrahydrofuran (20 mL) with triethylamine (0.4
mL 0.001 mol). The solution was cooled for 10 minutes in cold water. Ethyl chloroformate (0.2 mL
0.001 mol) was then added dropwise over a ten minute period. Once added, the cold water was
removed and the solution was stirred for 2 hours. After 2 hours, solvent removed under reduced
pressure then re-dissolved in dichloromethane and washed with water then 1-butylpiperazine (0.25 mL
0.001 mol) was added and the solution was stirred for 24 hours. Water (50 mL) was then added and
the solution extracted with ethyl acetate (3 x 50 mL). The organic layers were combined and washed
with saturated sodium hydrogen carbonate solution (3 x 50 mL). The organic layer was dried over
- 76 -
magnesium sulfate. Solvent was evaporated under reduced pressure. TLC EtOAc: MeOH 4:1 Rf 0.2
(single spot).
Yield; 0.04 g, 0.0004 mol, 6 %.
Melting point: 92.3 – 93.5 oC.
1H NMR (CDCl3) (250 MHz) δ= 0.68 (s, 3H, 18-CH3), 0.89 (s, 3H, 19-CH3), 0.98 (d, 3H, 21-CH3
J=7.5), 1.00-2.35 (m, 33H, steroidal backbone CH/CH2), 2.38 (m, 6H, CH2), 3.48 (broad singlet, 2H,
CH2), 3.62 (broad singlet, 2H, CH2), 3.98 (broad singlet, 1H, 12-CH) ppm.
13C NMR (CDCl3) (62.9 MHz) δ= 12.7 (CH3, C18), 14.0 (CH3 CH2CH3), 17.5 (CH3, C21), 20.6 (CH2),
23.1 (CH3, C19), 23.6 (CH2, C15), 26.1 (CH2), 27.5 (CH2, C7), 28.6 (CH2, C6), 28.8 (CH2, C16), 30.1
(CH2, C11), 30.5 (CH2, C2), 31.3 (CH2, C23), 33.6 (CH2, C22), 34.1 (CH, C9), 35.2 (C, C10), 35.2
(CH2, C1), 36.0 (CH, C20), 36.4 (CH2, C4), 41.4 (CH2), 42.0 (CH, C5), 45.6 (CH2), 46.5 (CH2), 47.2
(CH, C17), 48.3 (CH, C14), 52.8 (CH2), 53.4 (CH2), 71.8 (CH, C3), 73.1 (CH2, C12), 171.9 (CO, C24)
ppm.
IR= 3300 (OH), 2930 (alkyl), 2861 (alkyl), 1594 (C=O), 1443, 1282, 1162 (C=O ester stretch), 1011
(R2CH-OH) cm-1
.
MS (+ESI) m/z= Found 517.4359 (M+H)+; calculated for C32H57N2O3 517.4364; 0.9 ppm.
- 77 -
Preparation of (2S)-2-(tert-butoxycarbonylamino)-6-[[(4R)-4-[(3R,10S,13R,17R)-3-hydroxy-
10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-
17-yl]pentanoyl]amino]hexanoic acid (compound 68)
Lithocholic acid (0.5 g 0.001 mol) was dissolved in tetrahydrofuran (20 mL) with triethylamine (0.4
mL 0.002 mol). The solution was cooled for 10 minutes in cold water. Ethyl chloroformate (0.2 mL
0.001 mol) was then added dropwise over a ten minute period. Once added, the cold water was
removed and the solution was stirred for 2 hours. After 2 hours, solvent removed under reduced
pressure then re-dissolved in dichloromethane and washed with water then Boc-Lys-OH (0.32 g 0.001
mol) was added and the solution was stirred for 24 hours. Water (50 mL) was then added and the
solution extracted with ethyl acetate (3 x 50 mL). The crude material was purified by column
chromatography (9:1 chloroform/methanol). The organic layers were combined and dried over
magnesium sulfate. The solvent was removed under reduced pressure to produce viscous oil. TLC
EtOAc: MeOH 4:1 Rf 0.25 (single spot).
Yield; 0.19 g, 0.0003 mol, 24 %.
Melting point: Oil.
- 78 -
1H NMR (CDCl3 + D2O shake) (250 MHz) δ= 0.63 (s, 3H, 18-CH3), 0.91 (s, 3H, 19-CH3), 1.00-2.25
(m, 33H, steroidal backbone CH/CH2), 1.45 (s, 9H, BOC), 3.22 (broad s, 2H, CH2), 3.63 (m, 1H, 3-
CH), 4.25 (broad singlet, 1H, 12-CH) ppm.
13C NMR (CDCl3 + D2O shake) (62.9 MHz) δ= 12.0 (CH3, C18), 18.4 (CH3, C21), 20.8 (CH2, C11),
22.5 (CH2), 23.4 (CH3, C19), 24.2 (CH2, C15), 26.4 (CH2, C7), 27.2 (CH2, C6), 28.2 (CH2, C16), 28.3
(CH3 Boc), 28.9 (CH2), 29.3 (CH2), 30.2 (CH2, C2), 31.8 (CH2, C22), 32.3 (CH2, C23), 33.3 (C, C10),
34.5 (CH, C20), 35.3 (CH2, C1), 35.5 (CH, C8), 35.8 (CH2, C4), 36.1 (CH3), 39.2 (CH2, C12), 40.2
(CH, C9), 40.4 (CH, C5), 42.0 (C, C13), 42.7 (CH2), 53.4 (CH NHCHCOOH), 55.9 (CH2, C17), 56.5
(CH, C14), 71.8 (CH, C3), 155.0 (CH2), 174.7 (CO), 175.6 (CO, C24) ppm.
IR= 3343 (OH), 2921 (alkyl), 2857 (alkyl), 1705 (C=O), 1616, 1364, 1010 (R2CH-OH) cm-1
.
MS (+ESI) m/z= Found 603.4376 (M+H)+; calculated for C35H59N2O6 603.4379; 0.4 ppm
Preparation of (2S)-2-(tert-butoxycarbonylamino)-6-[[(4R)-4-[(3R,10S,12S,13R,17R)-3,12-
dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-
cyclopenta[a]phenanthren-17-yl]pentanoyl]amino]hexanoic acid (compound 69)
Deoxycholic acid (0.5 g 0.001 mol) was dissolved in tetrahydrofuran (15 mL) with triethylamine (0.20
mL 0.001 mol). The solution was cooled for 10 minutes in cold water. Ethyl chloroformate (0.2 mL
0.001 mol) was then added dropwise over a ten minute period. Once added, the cold water was
- 79 -
removed and the solution was stirred for 2 hours. After 2 hours Boc-Lys-OH (0.3 g 0.001 mol) was
added and the solution was stirred for a further 48 hours. Water (50 mL) was then added and the
solution extracted with ethyl acetate (3 x 50 mL). The organic layers were combined and washed with
saturated sodium hydrogen carbonate solution (3 x 50 mL). The crude material was purified by
column chromatography (9:1 chloroform/methanol). The organic layers were combined and dried over
magnesium sulfate. The solvent was removed under reduced pressure to produce a white solid. TLC
EtOAc: MeOH 4:1 Rf 0.25 (single spot).
Yield; 0.09 g, 0.0001 mol, 11 %.
Melting point: 97-100 oC.
1H NMR (CDCl3 + D2O shake) (250 MHz) δ= 0.67 (s, 3H, 18-CH3), 0.91 (s, 3H, 19-CH3), 0.99 (d, 3H,
21-CH3, J= 7.5), 1.00-2.17 (m, 33H, steroidal backbone CH/CH2), 1.45 (s, 9H, BOC), 3.24 (broad
singlet, 2H, CH2), 3.64 (m, 1H, 3-CH), 4.01 (s, 1H, 12-CH), 4.28 (broad singlet, 1H, CH) ppm.
13C NMR (CDCl3 + D2O shake) (62.9 MHz) δ= 12.6 (CH3, C18), 17.5 (CH3, C21), 22.3 (CH3, Boc),
23.0 (CH2), 23.7 (CH3, C19), 26.2 (CH2, C15), 27.1 (CH2, C7), 27.5 (CH2), 28.3 (CH2, C6), 28.7
(CH2, C16), 30.1 (CH2, C11), 31.5 (CH2, C2), 32.8 (CH2, C23), 33.5 (CH2, C22), 34.1 (CH, C9), 35.2
(C, C10), 35.3 (CH2, C1), 35.9 (CH, C20), 39.0 (CH2), 42.0 (CH, C5), 46.2 (C, C13), 46.4 (CH, C17),
48.2 (CH, C14), 53.4 (CH3), 71.8 (CH, C3), 73.4 (CH2, C12), 155.8 (COOH), 174.8 (CONH, C24)
ppm.
IR= 3334 (OH), 2933 (alkyl), 2865 (alkyl), 1697 (C=O), 1646 (C=O), 1530, 1360, 1159 (C=O ester
stretch), 1036 (R2CH-OH) cm-1
.
MS (+ESI) m/z= Found 619.4322 (M+H)+; calculated for C35H59N2O7 619.4328; 0.9 ppm.
- 80 -
Preparation of (4R)-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-
2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]-N-phenyl-
pentanamide (compound 70)
Based on the literature (Joachimiak et al., 2008) Lithocholic acid (2.0 g 0.005 mol) was dissolved in
tetrahydrofuran (60 mL) with triethylamine (0.64 mL, 0.006 mol). The solution was cooled for 10
minutes in cold water. Ethyl chloroformate (0.54 mL, 0.005 mol) was then added dropwise over a ten
minute period. Once added, the cold water was removed and the solution was stirred for 2 hours. After
2 hours aniline (0.54 mL, 0.005 mol) and DMAP (20 mg) were added and the solution was stirred for
a further 24 hours. Water (50 mL) was then added and the solution was extracted with ethyl acetate (3
x 50 mL). The organic layers were combined and washed with saturated sodium hydrogen carbonate
solution (3 x 50 mL). The organic layer was dried over magnesium sulfate. The solvent was
evaporated under reduced pressure and the product was recrystallized from ethyl acetate to produce a
white powder. TLC EtOAc: MeOH 4:1 Rf 0.2 (single spot).
Yield; 1.22 g, 0.002 mol, 51 %.
Melting point: 212.6-214.1 oC.
1H NMR (CDCl3) (250 MHz) δ= 0.65 (s, 3H, 18-CH3), 0.92 (s, 3H, 19-CH3), 1.01-2.50 (m, 33H,
steroidal backbone CH/CH2), 3.63 (m, 1H, 3-CH), 7.09 (t, 1H, Ar-CH, J= 5.0), 7.32 (t, 2H, Ar-CH, J =
7.5), 7.505 (d, 2H, Ar-CH, J= 7.5) ppm.
- 81 -
13C NMR (CDCl3) (62.9 MHz) δ= 12.0 (CH3, C18), 18.4 (CH3, C21), 20.8 (CH2, C11), 23.3 (CH3,
C19), 24.2 (CH2, C15), 26.4 (CH2, C7), 27.2 (CH2, C6), 28.2 (CH2, C16), 30.5 (CH2, C2), 31.6 (CH2,
C22), 34.5 (C, C10), 35.3 (CH, C20), 35.4 (CH2, C1), 35.8 (CH, C8), 36.4 (CH2, C4), 40.2 (CH2,
C12), 40.4 (CH, C9), 42.1 (CH, C5), 42.7 (C, C13), 55.0 (CH, C17), 56.5 (CH, C14), 71.8 (CH, C3),
119.7 (Ar-CH), 129.0 (Ar-CH), 171.6 (CO, C24) ppm.
IR= 3292 (OH), 2929 (alkyl), 2861 (alkyl), 1663 (C=O), 1539, 1437, 1309, 753 (aromatic) cm-1
.
MS (+ESI) m/z= Found 452.3519 (M+H)+; calculated for C30H46N2O2 452.3523; 0.9 ppm.
Preparation of (4R)-4-[(3R,10S,12S,13R,17R)-3,12-dihydroxy-10,13-dimethyl-
2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]-N-phenyl-
pentanamide (compound 71)
Deoxycholic acid (2.0 g 0.005 mol) was dissolved in tetrahydrofuran (60 mL) with N-
methylmorpholine (1.2 mL, 0.01 mol). The solution was cooled for 10 minutes in cold water. Ethyl
chloroformate (0.40 mL, 0.003 mol) was then added dropwise over a ten minute period. Once added,
the cold water was removed and the solution was stirred for 2 hours. After 2 hours aniline (0.52 mL,
0.005 mol) was added and the solution was stirred for a further 48 hours. Water (50 mL) was then
added and the resultant precipitate was collected by vacuum filtration. The product was recrystallized
from ethyl acetate to produce a white powder. TLC EtOAc: MeOH 4:1 Rf 0.25 (single spot).
Yield; 1.02 g, 0.002 mol, 42.8 %.
- 82 -
Melting point: 219.1 - 221.9 oC.
1H NMR (DMSO) (250 MHz) δ= 0.60 (s, 3H, 18-CH3), 0.85 (s, 3H, 19-CH3), 0.98-2.43 (m, 33H,
steroidal backbone CH/CH2), 3.81 (broad s, 1H, 12-CH), 4.19 (d, 1H, 3-OH, J= 2.5), 4.45 (d, 1H, 12-
OH, J= 5.0), 7.00 (t, 1H, Ar-CH, J= 7.5), 7.27 (t, 2H, Ar-CH, J= 7.5), 7.57 (d, 2H, Ar-CH, J= 7.5),
9.82 (s, 1H, NH) ppm.
13C NMR (DMSO) (62.9 MHz) δ= 12.4 (CH3, C18), 17.1 (CH3, C21), 23.0 (CH3, C19), 23.4 (CH2,
C15), 26.0 (CH2, C7), 26.9 (CH2, C6), 27.1 (CH2, C16), 30.2 (CH2, C11), 31.4 (CH2, C2), 32.9 (CH2,
C23), 33.4 (CH2, C22), 33.8 (CH, C9), 35.0 (C, C10), 35.6 (CH2, C1), 36.2 (CH, C20), 45.9 (CH, C5),
46.1 (C, C13), 47.4 (CH, C17), 69.9 (CH), 70.9 (CH, C3), 75.1 (CH2, C12), 118.9 (Ar-CH), 122.8 (Ar-
CH), 128.5 (Ar-CH), 139.3 (Ar-C), 171.6 (CO, C24) ppm.
IR= 3411 (OH), 2921 (alkyl), 2861 (alkyl), 1663 (C=O), 1590, 1548, 1441, 1249, 1040 (R2CH-OH)
cm-1
.
MS (+ESI) m/z= Found 468.3467 (M+H)+; calculated for C30H46NO3 468.3472; 1.1 ppm.
- 83 -
Preparation of (4R)-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-
2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]-N-(4-
vinylphenyl)pentanamide (compound 72)
Lithocholic acid (1.0 g 0.005 mol) was dissolved in tetrahydrofuran (30 mL) with triethylamine (0.64
mL 0.006 mol). The solution was cooled for 10 minutes in cold water. Ethyl chloroformate (0.19 mL,
0.0017 mol) was then added dropwise over a ten minute period. Once added, the cold water was
removed and the solution was stirred for 2 hours. After 2 hours 4-vinylaniline (0.37 mL 0.003 mol)
and 10 mg of 4-(dimethylamino)pyridine was added and the solution was stirred for a further 24 hours.
Water (50 mL) was then added and the solution extracted with ethyl acetate (3 x 50 mL). The organic
layers were combined and washed with hydrochloric acid (3 x 100 mL 3 mol). The organic layer was
dried over magnesium sulfate. Solvent was evaporated under reduced pressure to produce a white
powder. TLC EtOAc: MeOH 4:1 Rf 0.25 (single spot).
Yield; 0.27 g, 0.0005 mol, 20.61%
Melting point: 188.8 – 193.9 oC.
1H NMR (CDCl3) (250 MHz) δ= 0.65 (s, 3H, 18-CH3), 0.92 (s, 3H, 19-CH3), 1.03-2.50 (m, 33H,
steroidal backbone CH/CH2), 3.63 (m, 1H 3-CH), 5.19 (d, 1H =CH- J= 10.0), 5.67 (d, 1H, CH, J=
17.5), 6.67 (dd, 1H, CH, J= 12.5 and 22.5), 7.11 (Broad s, 1H, NH), 7.365 (d, 2H, Ar-CH, J= 7.5),
7.485 (d, 2H, Ar-CH, J= 7.5) ppm.
- 84 -
13C NMR (CDCl3) (62.9 MHz) δ= 12.0 (CH3, C18), 18.4 (CH3, C21), 20.8 (CH2, C11), 23.3 (CH3,
C19), 24.2 (CH2, C15), 26.4 (CH2, C7), 27.2 (CH2, C6), 28.2 (CH2, C16), 34.5 (C, C10), 34.6 (CH,
C20), 35.3 (CH2, C1), 35.4 (CH, C8), 35.8 (CH2, C4), 40.2 (CH2, C12), 40.4 (CH, C9), 42.0 (CH, C5),
42.7 (C, C13), 56.0 (CH, C17), 56.5 (CH, C14), 71.8 (CH, C3), 119.5 (Ar-CH), 120.6 (Ar-CH), 136.0
(Ar-CH), 144.0 (Ar-C) ppm.
IR= 3449 (OH), 2925 (alkyl), 2857 (alkyl), 1667 (C=O), 1513, 1313, 1245, 1027 (R2CH-OH) cm-1
.
MS (+ESI) m/z= Found 478.3671 (M+H)+; calculated for C32H48NO2 478.3680; 1.8 ppm.
Preparation of (4R)-4-[(3R,10S,12S,13R,17R)-3,12-dihydroxy-10,13-dimethyl-
2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]-N-(4-
vinylphenyl)pentanamide (compound 73)
Deoxycholic acid (2.0 g 0.005 mol) was dissolved in 1, 4 dioxane (60 mL) with triethylamine (1.32
mL, 0.01 mol). The solution was cooled for 10 minutes in cold water. Ethyl chloroformate (0.4 mL,
0.003 mol) was then added dropwise over a ten minute period. Once added, the cold water was
removed and the solution was stirred for 2 hours. After 1 hour, 4-vinylaniline (0.31 g, 0.002 mol) and
DMAP (10 mg) were added and the solution was stirred for a further 48 hours. Water (50 mL) was
then added and the resultant precipitate was collected by vacuum filtration. This product was re-
dissolved in ethyl acetate and washed with 3 M HCl (3 x 100 mL). The organic layer was dried over
- 85 -
magnesium sulfate. The solvent was evaporated under reduced pressure to produce a white powder.
TLC EtOAc: MeOH 4:1 Rf 0.25 (single spot).
Yield not recorded.
Melting point: 98.7-101.5 oC.
1H NMR (CDCl3) (250 MHz) δ= 0.68 (s, 3H, 18-CH3), 0.91 (s, 3H, 19-CH3), 1.00-2.38 (m, 33H,
steroidal backbone CH/CH2), 3.62 (m, 1H, 3-CH), 3.99 (broad singlet, 1H, 12-CH), 5.185 (d, 2H,
=CH-, J= 12.5), 5.675 (d, 2H, CH, J= 17.5), 6.675 (dd, 1H, CH, J values = 10 and 17.5), 7.355 (d, 2H,
Ar-CH, J= 7.5), 7.505 (d, 2H, Ar-CH, J= 7.5) ppm.
13C NMR (CDCl3) (62.9 MHz) δ= 12.7 (CH3, C18), 17.5 (CH3, C21), 23.1 (CH2), 23.7 (CH3, C19),
26.1 (CH2, C15), 27.1 (CH2, C7), 27.5 (CH2, C6), 28.5 (CH2, C16), 30.5 (CH2, C2), 31.3 (CH2, C23),
33.6 (CH2, C22), 34.1 (CH, C9), 35.1 (C, C10), 36.0 (CH2, C1), 36.4, 42.0 (CH, C5), 46.5 (C, C13),
46.9 (CH, C17), 48.2 (CH, C14), 71.8 (CH, C3), 112.8 (Ar-CH), 119.66 (Ar or vinyl CH), 126.8 (Ar
or vinyl CH), 133.5 (Ar-C), 136.1 (Ar or vinyl-CH), 137.7 (Ar-C), 171.9 (CO, C24) ppm.
IR= 3300 (OH), 2925 (alkyl), 2861 (alkyl), 1663 (C=O), 1599, 1245, 1040 (R2CH-OH) cm-1
.
MS (+ESI) m/z= Found 494.3623 (M+H)+; calculated for C32H48N1O3 494.3629; 1.2 ppm.
- 86 -
Preparation of methyl 4-[[(4R)-4-[(3R,10S,12S,13R,17R)-3,12-dihydroxy-10,13-dimethyl-
2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-
yl]pentanoyl]amino]benzoate (compound 74)
Deoxycholic acid (2.0 g 0.005 mol) was dissolved in 1, 4-dioxane (60 mL) with triethylamine (1.32
mL 0.01 mol). The solution was cooled for 10 minutes in cold water. Ethyl chloroformate (0.40 mL
0.003 mol) was then added dropwise over a ten minute period. Once added, the cold water was
removed and the solution was stirred for 2 hours. After 2 hours methyl 4-aminobenzoate (0.84 g 0.005
mol) was added and the solution was stirred for a further 24 hours. Water (50 mL) was then added and
the solution extracted with ethyl acetate (3 x 50 mL). The organic layers were combined and washed
with saturated sodium hydrogen carbonate solution (3 x 50 mL). The organic layer was dried over
magnesium sulfate. Solvent was evaporated under reduced pressure to produce a white powder. TLC
EtOAc: MeOH 4:1 Rf 0.25 (single spot).
Yield; 0.57 g, 0.001 mol, 21 %.
Melting point: 124.3-127.6 oC.
1H NMR (250 MHz) (DMSO) δ= 0.60 (s, 3H, 18-CH3), 0.85 (s, 3H, 19-CH3), 1.00-2.38 (m, 33H,
steroidal backbone CH/CH2), 3.81 (s, 3H, O-CH3), 4.20 (d, 1H, 3-OH, J= 5.0), 4.45 (d, 1H, 12-OH, J=
5.0), 7.72 (d, 2H, Ar-CH, J= 10.0), 7.895 (d, 2H, Ar-CH, J= 7.5), 10.20 (broad singlet, 1H, NH) ppm.
- 87 -
13C NMR (62.9 MHz) (MeOD) δ= 13.2 (CH3, C18), 17.7 (CH3, C21), 23.7 (CH3, C19), 24.9 (CH2,
C15), 27.5 (CH2, C7), 28.4 (CH2, C6), 28.7 (CH2, C16), 29.9 (CH2, C11), 33.0 (CH2, C22), 34.8 (CH,
C9), 35.1 (C, C10), 35.3 (CH2, C1), 36.4 (CH, C20), 36.9 (CH2, C4), 37.2 (CH, C8), 37.5 (CH3 O-
CH3), 43.6 (CH, C5), 52.4 (CH2), 72.5 (CH, C3), 74.0 (CH2, C12), 120.1 (Ar-CH), 126.2 (Ar-CH),
131.5 (Ar-CH), 144.7 (Ar-C), 168.2 (COOCH) 175.5 (CONH, C24) ppm.
IR= 3478 (OH), 2935 (alkyl), 2865 (alkyl), 1691, 1669 (C=O), 1593, 1536, 1430, 1275, 1175 (C-O
ester stretch), 1038 (R2CH-OH) cm-1
.
MS (+ESI) m/z= Found 526.3522 (M+H)+; calculated for C32H48N1O5 526.3527; 1 ppm.
4-[(3R,10S,13R,17R)-3-Hydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-
tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]-N-(2-pyrrolidin-1-ylethyl)pentanamide
(compound 75)
Lithocholic acid (0.5 g 0.001 mol) was dissolved in tetrahydrofuran (20 mL) with triethylamine (0.20
mL, 0.001 mol). The solution was cooled for 10 minutes in cold water. Ethyl chloroformate (0.20 mL,
0.001 mol) was then added dropwise over a ten minute period. Once added, the cold water was
removed and the solution was stirred for 2 hours at room temperature. After 2 hours 1-(2-
aminoethyl)pyrollidine (0.25 mL, 0.002 mol) was added and the solution was stirred for 3 hours.
Water (50 mL) was then added and the solution was extracted with ethyl acetate (3 x 50 mL). The
- 88 -
organic layers were combined and dried over magnesium sulfate. The solvent was evaporated under
reduced pressure to produce a white powder. TLC EtOAc: MeOH 4:1 Rf 0.25 (single spot).
Yield; 0.56 g, 0.001 mol, 91.8 %.
Melting point: 144.1- 145.1 oC.
1H NMR (CDCl3) (250 MHz) δ= 0.64 (s, 3H, 18-CH3), 0.92 (s, 3H, 19-CH3), 0.94 (d, 3H, 21-CH3, J=
7.5), 1.00-2.26 (m, 33H, steroidal backbone CH/CH2), 1.80 (m, 4H, CH2), 2.54 (broad singlet, 4H,
CH2), 2.60 (t, 2H, CH2, J=5.0), 3.365 (q, 2H, CH2, J= 7.5), 3.63 (m, 1H, 3-CH), 6.13 (broad s, 1H,
NH) ppm.
13C NMR (CDCl3) (62.9 MHz) δ= 12.0 (CH3, C18), 18.4 (CH3, C21), 20.8 (CH2, C11), 23.4 (CH3,
C19), 24.2 (CH2, C15), 26.4 (CH2, C7), 27.2 (C H2, C6), 28.2 (CH2, C16), 30.5 (CH2, C2), 31.7 (CH2,
C22), 33.5 (CH2, C23), 34.5 (C, C10), 35.3 (CH, C20), 35.5 (CH2, C1), 35.8 (CH, C8), 36.4 (CH2,
C4), 37.7 (CH2), 40.2 (CH2, C12), 40.4 (CH, C9), 42.1 (CH, C5), 42.7 (C, C13), 53.8 (CH2, NH-CH2
or CH2N(CH3)2), 54.9 (CH2, NH-CH2 or CH2N(CH3)2), 56.0 (CH, C17), 56.5 (CH, C14), 71.8 (CH,
C3), 173.9 (CO, C24) ppm.
IR= 3415 (NH), 3310 (OH), 2933 (alkyl), 2865 (alkyl), 2872 (alkyl), 1648 (C=O), 1548, 1444, 1378,
1265 (C-O ester stretch), 1064 (R2CH-OH) cm-1
.
MS (+ESI) m/z= Found 473.4096 (M+H)+; calculated for C30H53N2O2 473.4102; 1.2 ppm.
- 89 -
Preparation of 4-[(3R,10S,12S,13R,17R)-3,12-dihydroxy-10,13-dimethyl-
2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]-N-(2-
pyrrolidin-1-ylethyl)pentanamide (compound 76)
Deoxycholic acid (2.0 g 0.005 mol) was dissolved in 1, 4-dioxane (60 mL) with triethylamine (1.28
mL, 0.01 mol). The solution was cooled for 10 minutes in cold water. Ethyl chloroformate (0.37 mL,
0.003 mol) was then added dropwise over a ten minute period. Once added, the cold water was
removed and the solution was stirred for 2 hours. After 2 hours 1-(2-aminoethyl) pyrrolidine (0.77 mL,
0.006 mol) was added and the solution was stirred for 3 hours at room temperature. Water (50 mL)
was then added and the solution was extracted with ethyl acetate (3 x 50 mL). The organic layers were
combined and washed with saturated sodium hydrogen carbonate solution (3 x 50 mL). The organic
layer was dried over magnesium sulfate. The solvent was evaporated under reduced pressure to
produce a white powder. TLC EtOAc: MeOH 4:1 Rf 0.2 (single spot).
Yield; 1.2 g, 0.0025 mol, 49 %.
Melting point: 155.3-158.7 oC.
1H NMR (CDCl3) (250 MHz) δ= 0.66 (s, 3H, 18-CH3), 0.89 (s, 3H, 19-CH3), 0.975 (d, 3H, 21-CH3, J=
7.5), 1.00-2.30 (m, 33H, steroidal backbone CH/CH2), 1.78 (m, 4H, CH2), 2.42-2.58 (m, 6H, CH2),
3.35 (m, 2H, CH2), 3.58 (m, 1H, 3-CH), 3.95 (broad s, 1H, 12-CH), 6.49 (broad s, 1H, NH) ppm.
- 90 -
13C NMR (CDCl3) (62.9 MHz) δ= 12.7 (CH3, C18), 17.5 (CH3, C21), 23.3 (CH3, C19), 23.7 (CH2,
C15), 26.1 (CH2, C7), 27.1 (CH2, C6), 28.6 (CH2, C16), 30.4 (CH2, C11), 30.5 (CH2, C2), 33.3 (CH2,
C23), 33.6 (CH2, C22), 34.1 (CH, C9), 35.2 (C, C10), 35.3 (CH2, C1), 36.0 (CH, C20), 36.5 (CH2,
C4), 37.9 (CH, C8), 42.1 (CH2), 46.5 (C, C13), 46.8 (CH2, C17), 48.2 (CH2, C14), 53.9 (CH2), 55.0
(CH2), 71.5 (CH, C3), 73.0 (CH2, C12), 173.9 (CO, C24) ppm.
IR= 3287 (OH), 2916 (alkyl), 2865 (alkyl), 1641 (C=O), 1539, 1441, 1040 (R2CH-OH) cm-1
.
MS (+ESI) m/z= Found 489.4046 (M+H)+; calculated for C30H53N2O3 489.4051; 1 ppm.
Preparation of 4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-
tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]-N-[2-(1-piperidyl)ethyl]pentanamide
(compound 77)
From a modification of a method.(Liu et al., 2001) Methyl lithocholate 0.5g (0.001mol) 1,2 amino-
ethyl piperidine 0.36 mL (0.002mol) were heated at and stirred at 150°C for 48 hours in a argon
environment. TLC (Ethyl acetate: Methanol 4:1) indicated partial consumption of starting material so
another 1 mL (0.007mol) of 1,2 amino-ethyl piperidine was added and the reaction was put on with
the same conditions overnight. TLC indicated that starting material had been consumed but that the
mixture was not pure. Purified by column chromatography (1:1 EtOAc/MeOH).
Yield; 0.01 g, 0.00001 mol, 1.6%.
- 91 -
Melting point: 85.0-89.9 °C.
1H NMR (CDCl3) (250 MHz) δ= 0.59 (s, 3H, 18-CH3), 0.87-0.89 (s, 3H, 19-CH3 J=5.5), 1.00-2.38 (m,
33H, steroidal backbone CH/CH2), 1.72 (m, 4H, CH2), 2.68 (t, 2H, CH2, J=5.6), 3.42-3.44 (q, 2H, CH2,
J=5.6), 3.58 (m, 1H, 3-CH), 7.18 (s/t, 1H, NH) ppm.
13C NMR (62.9 MHz) δ= 12.0 (CH3, C18), 18.4 (CH3, C21), 20.8 (CH2, C11), 23.4 (CH3,
C19), 24.2 (CH2, C15), 24.3 (CH2), 25.9 (CH2), 26.4 (CH2, C7), 27.2 (CH2, C6), 28.2 (CH2,
C16), 30.5 (CH2, C2), 31.8 (CH2, C22), 33.6 (CH2, C23), 34.5 (C, C10), 35.34 (CH, C20),
35.5 (CH2, C1), 35.8 (CH, C8), 35.9 (CH2, C4), 36.45 (Unknown C), 40.1 (CH2, C12), 40.4
(CH, C9), 42.0 (CH, C5), 42.7 (C, C13), 54.2 (CH, C17), 56.0 (CH, C14), 56.4 (CH2), 57.2
(CH2), 71.8 (CH, C3), 173.6 (CO, C24) ppm.
IR= 3418 (NH), 3465 (OH) 2935 (alkyl), 2865 (alkyl), 1641 (C=O) cm-1
.
MS (ES) m/z= Found 487.4347 (M+H)+; calculated for C31H54N2O3 487.4264; 0.8 ppm.
Preparation of (4R)-4-[(3R,10S,12S,13R,17R)-3,12-dihydroxy-10,13-dimethyl-
2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]-N-[2-(1-
piperidyl)ethyl]pentanamide (compound 78)
Methyl deoxycholate (1.0 g 0.002 mol) was dissolved in methanol (10 mL) with 1-(2-
aminoethyl)piperidine (3.15 g, 0.024 mol) was added and the solution was stirred for 5 days at reflux.
The solvent was evaporated under reduced pressure and the product was recrystallized from ethyl
acetate to produce a white powder. TLC EtOAc: MeOH 4:1 Rf 0.25 (single spot).
- 92 -
Yield; 0.56 g, 0.0011 mol, 45 %.
Melting point: 158.5-161.1 oC.
1H NMR (CDCl3) (250 MHz) δ= 0.66 (s, 3H, 18-CH3), 0.90 (s, 3H, 19-CH3), 0.99 (d, 3H, 21-CH3, J=
5.0), 1.00-2.38 (m, 33H, steroidal backbone CH/CH2), 2.52 (m, 2H, CH2), 3.385 (q, 2H, CH2, J= 5.0),
3.60 (m, 1H, 3-CH), 3.97 (s, 1H, 12-CH), 6.72 (broad s, 1H, NH) ppm.
13C NMR (CDCl3) (62.9 MHz) δ= 12.7 (CH3, C18), 17.5 (CH3, C21), 23.1 (CH3, C19), 23.7 (CH2),
23.8 (CH2), 25.2 (CH2, C15), 26.1 (CH2) 27.1 (CH2, C7), 27.5 (CH2, C6), 28.6 (CH2, C16), 30.5 (CH2,
C2), 31.6 (CH2, C23), 33.3 (CH2, C22), 33.6 (CH, C9), 34.1 (C, C10), 35.2 (CH2, C1), 35.2 (CH,
C20), 35.4 (CH2, C4), 36.0 (CH, C8), 36.5 (CH, C5), 42.0 (C, C13), 46.5 (CH, C17), 47.0 (CH2), 48.3
(CH, C14), 54.2 (CH2), 57.4 (CH2), 71.6 (CH, C3), 73.0 (CH2, C12), 173.9 (CO, C24) ppm.
IR= 3300 (OH), 2925 (alkyl), 2861 (alkyl), 1642 (C=O), 1543, 1437, 1313, 1040 (R2CH-OH) cm-1
.
MS (+ESI) m/z= Found 503.4202 (M+H)+; calculated for C31H55N2O3 503.4207; 1 ppm.
Preparation of (4R)-N-(4-benzoylphenyl)-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-
2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-
yl]pentanamide (compound 79)
Lithocholic acid (2.0 g 0.005 mol) was dissolved in tetrahydrofuran (60 mL) with N-
methylmorpholine (1.07 mL, 0.01 mol). The solution was cooled for 10 minutes in cold water. Ethyl
- 93 -
chloroformate (0.54 mL, 0.005 mol) was then added dropwise over a ten minute period. Once added,
the cold water was removed and the solution was stirred for 2 hours. After 2 hours amino-
benzophenone (1.5 g, 0.007 mol) was added and the solution was stirred for a further 48 hours. Water
(50 mL) was then added and the solution was extracted with ethyl acetate (3 x 50 mL). The organic
layers were combined and washed with saturated sodium hydrogen carbonate solution (3 x 50 mL) and
2 M hydrochloric acid solution (3 x 50 mL). The organic layer was dried over magnesium sulfate. The
solvent was evaporated under reduced pressure and the product was triturated with ethyl acetate to
produce a white powder. TLC EtOAc: MeOH 4:1 Rf 0.25 (single spot).
Yield 0.57g, 0.001 mol, 19 %.
Melting point: 228.6-230.8 oC.
1H NMR (d6 DMSO) (250 MHz) δ= 0.62 (s, 3H, 18-CH3), 0.87 (s, 3H, 19-CH3), 0.92 (d, 2H, CH2 J=
7.5), 1.00-2.50 (m, 33H, steroidal backbone CH/CH2), 4.44 (d, 1H, 3-OH, J= 5.0), 7.52-7.805 (m, 9H,
Ar-CH), 10.20 (s, 1H, NH) ppm.
13C NMR (d6 DMSO) (62.9 MHz) δ= 11.8 (CH3, C18), 18.3 (CH3, C21), 20.3 (CH2, C11), 23.2 (CH3,
C19), 23.8 (CH2, C15), 26.1 (CH2, C7), 26.8 (CH2, C6), 27.7 (CH2, C16), 30.3 (CH2, C2), 31.1 (CH2,
C22), 33.4 (CH2, C23), 34.1 (C, C10), 34.9 (CH, C20), 35.1 (CH2, C1), 35.3 (CH, C8), 41.4 (CH, C5),
42.2 (C, C13), 55.5 (CH, C17), 56.0 (CH, C14), 69.8 (CH, C3), 118.1 (Ar-CH), 128.4 (Ar-CH), 129.3
(Ar-CH), 131.0 (Ar-CH), 131.1 (Ar-CH), 132.1 (Ar-CH), 137.5 (Ar-C), 143.5 (Ar-C), 172.3 (CO,
C24), 194.4 (Ar-CO) ppm.
IR= 3488 (NH), 3249 (OH), 2925 (alkyl), 2857 (alkyl), 1675 (C=O), 1586, 1296, 1245, 1168, 1031
(R2CH-OH) cm-1
.
MS (+ESI) m/z= Found 556.3781 (M+H)+; calculated for C37H50NO3 556.3785; 0.8 ppm.
- 94 -
Preparation of (4R)-N-(4-benzoylphenyl)-4-[(3R,10S,12S,13R,17R)-3,12-dihydroxy-10,13-
dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-
yl]pentanamide (compound 80)
Deoxycholic acid (2.0 g 0.005 mol) was dissolved in 1, 4-dioxane (60 mL) with N-methylmorpholine
(1.07 mL 0.01 mol). The solution was cooled for 10 minutes in cold water. Ethyl chloroformate (0.69
mL 0.006 mol) was then added dropwise over a ten minute period. Once added, the cold water was
removed and the solution was stirred for 2 hours. After 2 hours amino-benzophenone (1.5 g 0.007
mol) was added and the solution was stirred for a further 24 hours. Water (50 mL) was then added and
the solution extracted with ethyl acetate (3 x 50 mL). The organic layers were combined and washed
with saturated sodium hydrogen carbonate solution (3 x 50 mL) and 2 M hydrochloric acid (3 x 50
mL). The organic layer was dried over magnesium sulfate. Solvent was evaporated under reduced
pressure and triturated with ethyl acetate to produce a white powder. TLC EtOAc: MeOH 4:1 Rf 0.25
(single spot).
Yield; 0.24 g, 0.0004 mol, 8 %.
Melting point: 220-227.6 oC.
1H NMR (d6 DMSO) (250 MHz) δ= 0.60 (s, 3H, 18-CH3), 0.85 (s, 3H, 19-CH3), 0.97 (d, 3H, 21-CH3,
J= 5.0), 1.00-2.50 (m, 33H, steroidal backbone CH/CH2), 3.80 (s, 1H, 3-CH), 4.03 (s, 1H, 12-CH),
- 95 -
4.21 (d, 1H, 3-OH, J= 2.5), 4.46 (d, 1H, 12-OH, J= 5.0), 7.52-7.79 (multiple overlapping multiplets,
9H, Ar-CH), 10.26 (s, 1H, NH) ppm.
13C NMR (d6 DMSO) (62.9 MHz) δ= 12.4 (CH3, C18), 17.0 (CH3, C21), 23.0 (CH3, C19), 26.9 (CH2,
C7), 27.2, (CH2, C2), 32.9 (CH2, C22), 33.5 (CH, C9), 33.7 (C, C10), 35.0 (CH2, C1), 35.6 (CH, C20),
45.9 (C, C13), 46.1 (CH, C17), 47.2 (CH, C14), 70.9 (CH, C3), 118.1 (Ar-CH), 128.4 (Ar-CH), 129.3
(Ar-CH), 131.1 (Ar-CH), 132.1 (Ar-CH), 137.5 (Ar-C), 143.5 (Ar-C), 172.4 (CONH, C24), 194.5 (Ar-
CO) ppm.
IR= 3462 (OH), 2921 (alkyl), 2861 (alkyl), 1675 (C=O), 1586 (C=O), 1441, 1279, 1168.78 (C-O ester
stretch), 1036 (R2CH-OH) cm-1
.
MS (+ESI) m/z= Found 572.3730 (M+H)+; calculated for C37H50NO4 572.3734; 0.8 ppm.
Preparation of (4R)-N-(4-benzoylphenyl)-4-[(3R,7R,10S,12S,13R,17R)-3,7,12-trihydroxy-10,13-
dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-
yl]pentanamide (compound 81)
Cholic acid (2.0 g 0.004 mol) was dissolved in 1, 4-dioxane (60 mL) with N-methylmorpholine (1.07
mL 0.01 mol). The solution was cooled for 10 minutes in cold water. Ethyl chloroformate (0.66 mL
0.006 mol) was then added dropwise over a ten minute period. Once added, the cold water was
removed and the solution was stirred for 2 hours. After 2 hours amino-benzophenone (1.44 g 0.007
- 96 -
mol) was added and the solution was stirred for a further 48 hours. Water (50 mL) was then added and
the solution extracted with ethyl acetate (3 x 50 mL). The organic layers were combined and washed
with saturated sodium hydrogen carbonate solution (3 x 50 mL) and 2 M hydrochloric acid (3 x 50
mL). The organic layer was dried over magnesium sulfate. Solvent was evaporated under reduced
pressure and triturated with ethyl acetate to produce a white powder. TLC EtOAc: MeOH 4:1 Rf 0.25
(single spot).
Yield; 0.77 g, 0.001 mol, 31 %.
Melting point: 250.5-254.8 oC.
1H NMR (d6 DMSO) (250 MHz) δ= 0.61 (s, 3H, 18-CH3), 0.82 (s, 3H, 19-CH3), 0.995 (d, 2H, CH2
J= 7.5), 1.31-2.50 (m, 33H, steroidal backbone CH/CH2), 3.2, 3.63, 3.81 (broad s, 3H, 7, 3, 12-CH),
4.02, 4.13, 4.34 (d, 3H, 7, 3, 12-OH, J= 5.0), 7.56-7.77 (m, 9H, Ar-CH), 10.27 (s, 1H, NH) ppm.
13C NMR (d6 DMSO) (62.9 MHz) δ= 12.3 (CH3, C18), 17.1 (CH3, C21), 22.6 (CH3, C19), 22.7 (CH2,
C15), 26.1 (CH, C9), 27.2 (CH2, C16), 28.5 (CH2, C11), 30.3 (CH2, C3), 31.2 (CH2, C23), 33.5 (CH2,
C22), 34.3 (CH2, C6), 34.8 (C, C10), 35.1 (CH2, C1), 35.2 (CH, C20), 41.3 (CH, C8), 41.4 (CH, C14),
45.7 (CH, C5), 46.0 (C, C13), 66.2 (CH, C7), 66.3 (CH, C3), 70.4 (CH), 70.9 (CH, C12), 118.1 (Ar-
CH), 128.4 (Ar-CH), 129.3 (Ar-CH), 130.9 (Ar-CH), 131.1 (Ar-CH), 137.5 (Ar-C), 143.5 (Ar-C),
172.4 (C, CONH), 194.4 (Ar-CO) ppm.
IR= 3462 (OH), 2933 (alkyl), 2870 (alkyl), 1671 (C=O), 1594, 1164, 1279, 1091 (R2CH-OH) cm-1
.
MS (+ESI) m/z= Found 588.3680 (M+H)+; calculated for C37H50NO5 588.3684; 0.6 ppm.
- 97 -
Preparation of (4R)-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-
2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]-N-[4-
[hydroxy(phenyl)methyl]phenyl]pentanamide (compound 82)
Compound 22 (0.25 g, 0.0006 mol) was dissolved in tetrahydrofuran (10 mL) with sodium
borohydride (0.34 g, 0.008 mol). The solution was stirred for 2 days at room temperature. Water (10
mL) was then added and the precipitate collected by vacuum filtration, washed with water (3 x 20 mL)
and dried under vacuum. TLC EtOAc: MeOH 4:1 Rf 0.25 (single spot).
Yield 0.18 g, 0.003 mol. 72 %.
Melting point: 211.2 – 217.6 oC.
1H NMR (d6 DMSO) (250 MHz) δ= 0.61 (s, 3H, 18-CH3), 0.87 (s, 3H, 19-CH3), 0.92 (d, 3H, 21-CH3
J= 7.5), 1.00-2.38 (m, 33H, steroidal backbone CH/CH2), 4.43 (s, 1H, 3-OH), 5.62 (s, 1H, CH-O), 5.80
(broad s, 1H, OH), 7.27-7.47 (m, 9H, Ar-CH), 9.79 (s, 1H, CH) ppm.
13C NMR (d6 DMSO) (62.9 MHz) δ= 11.8 (CH3, C18), 18.3 (CH3, C21), 20.3 (CH2, C11), 23.2 (CH3,
C19), 23.8 (CH2, C15), 26.1 (CH2, C7), 27.7 (CH2, C6), 30.3 (CH2, C2), 31.3 (CH2, C22), 34.1 (C,
C10), 34.9 (CH, C20), 35.3 (CH2, C1), 41.4 (CH, C5), 42.2 (C, C13), 55.5 (CH, C17), 56.0 (CH, C14),
69.8 (CH, C3), 73.8 (CH2), 79.1 (CH2), 118.7 (Ar-CH), 126.1 (Ar-CH), 126.4 (Ar-CH), 127.9 (Ar-
CH), 137.9 (Ar-C), 140.2 (Ar-CH), 145.7 (Ar-C), 171.4 (CO, C24) ppm.
- 98 -
IR= 3424 (NH), 2925 (alkyl), 2861 (alkyl), 1663 (C=O), 1599, 1535, 1407, 1300, 1031 (R2CH-OH)
cm-1
.
MS (+ESI) m/z= Found 575.4207 (M + NH4); calculated for C37H55N2O3 575.4207; 0.6 ppm.
Preparation of (4R)-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-
2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]-1-[4-(4-
pyridyl)piperazin-1-yl]pentan-1-one (compound 83).
From a modification (El Kihel et al., 2008), lithocholic acid (0.5 g, 0.001 mol) was dissolved in
tetrahydrofuran (15 mL) with triethylamine (0.20 mL, 0.001 mol). The solution was cooled for 10
minutes in cold water. Ethyl chloroformate (0.2 mL, 0.001 mol) was then added dropwise over a ten
minute period. Once added, the cold water was removed and the solution was stirred for 2 hours. After
2 hours 1-(4-pyridyl)piperazine (0.21 mL, 0.001 mol) was added and the solution was stirred for 24
hours. Water (50 mL) was then added and the solution extracted with ethyl acetate (3 x 50 mL). The
organic layers were combined and washed with saturated sodium hydrogen carbonate solution (3 x 50
mL). Due to impurities, re-dissolved in a methanol/water solution (20 mL 50/50) and extracted with
chloroform (4 x 20 mL). The organic layer was dried over magnesium sulfate. Solvent was evaporated
under reduced pressure to produce a white powder. TLC EtOAc: MeOH 4:1 Rf 0.25 (single spot).
Yield; 0.03 g, 0.00005 mol, 4 %.
Melting point: 150-157 oC.
- 99 -
1H NMR (CDCl3) (250 MHz) δ= 0.65 (s, 3H, 18-CH3), 0.92 (s, 3H, 19-CH3), 1.00-2.40 (m, 33H,
steroidal backbone CH/CH2), 3.35 (m, 4H, CH2), 3.64 (m, 2H, CH2), 6.68 (d, 2H, Ar-CH, J = 5.0),
8.31 (s, 2H, Ar-CH) ppm.
13C NMR (62.9 MHz) δ= 12.0 (CH3, C18), 18.6 (CH3, C21), 20.8 (CH2, C11), 23.3 (CH3, C19), 24.3
(CH2, C15), 26.3 (CH2, C7), 27.1 (CH2, C6), 28.3 (CH2, C16), 30.2 (CH2, C2), 31.3 (CH2, C22), 34.5
(C, C10), 35.4 (CH, C20), 35.8 (CH2, C1), 36.4 (CH2, C4), 40.1 (CH2, C12), 40.4 (CH, C9), 40.7 (CH,
C5), 42.1 (C, C13), 42.7 (CH2 N-CH2), 44.7 (CH2, N-CH2), 45.8 (CH2, N-CH2), 45.9 (CH2, N-CH2),
55.9 (CH, C17), 56.5 (CH, C14), 71.8 (CH, C3), 108.5 (Ar-CH), 129.7 (Ar-CH), 149.9 (Ar-C), 150.0
(Ar-CH), 154.6 (Ar-CH), 172.7 (CO, C24) ppm.
IR= 3387 (OH), 2925 (alkyl), 2852 (alkyl), 1641 (C=O), 1445, 1236, 1044, 989 (R2CH-OH) cm-1
.
MS (ES) m/z= Found 522.4048 (M+H)+ ; calculated for C31H57N2O4 521.4313; 1.5 ppm.
Preparation of 2-[[(4R)-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-
2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-
yl]pentanoyl]amino]benzamide (compound 84)
Lithocholic acid (2.0 g 0.005 mol) was dissolved in 1, 4 dioxane (40 mL) with triethylamine (1.40 mL,
0.01 mol). The solution was cooled for 10 minutes in cold water. Ethyl chloroformate (0.54 mL, 0.005
mol) was then added dropwise over a ten minute period. Once added, the cold water was removed and
the solution was stirred for 2 hours. After 2 hours 2-aminobenzamide (0.7 g, 0.005 mol) was added
and the solution was stirred for a further 48 hours. Water (50 mL) was then added and the solution was
- 100 -
extracted with ethyl acetate (3 x 50 mL). The organic layers were combined and washed with saturated
sodium hydrogen carbonate solution (3 x 50 mL). The organic layer was dried over magnesium
sulfate. The solvent was evaporated under reduced pressure and the product was recrystallized from
ethyl acetate to produce a white powder. TLC EtOAc: MeOH 4:1 Rf 0.25 (single spot).
Yield; 1.41 g, 0.002 mol, 53 %.
Melting point: 112.1-113.0 oC.
1H NMR (CDCl3) (250 MHz) δ= 0.63 (s, 3H, 18-CH3), 0.90 (s, 3H, 19-CH3), 1.0-2.50 (m, 33H,
steroidal backbone CH/CH2), 3.63, (m, 1H, 3-CH), 5.92 (broad singlet, 1H, split NH2), 6.39 (broad
singlet, 1H, split NH2), 7.05 (t, 1H, Ar-CH), 7.51 (t, 1H, Ar-CH), 8.625 (d, 1H, Ar-CH, J= 7.5), 11.15
(s, 1H, NH) ppm.
13C NMR (CDCl3) (62.9 MHz) δ= 12.0 (CH3, C18), 18.4 (CH3, C21), 20.8 (CH2, C11), 23.4 (CH3,
C19), 24.2 (CH2, C15), 26.4 (CH2, C7), 27.2 (CH2, C6), 28.2 (CH2, C16), 30.5 (CH2, C23), 34.5 (C,
C10), 35.3 (CH, C20), 35.4 (CH2, C1), 35.4 (CH, C8), 35.8 (CH2, C4), 36.4 (CH2), 40.1 (CH2, C12),
40.4 (CH, C9), 42.1 (CH, C5), 42.7 (C, C13), 55.9 (CH, C17), 56.4 (CH, C14), 71.8 (CH, C3), 118.4
(Ar-C), 121.5 (Ar-CH), 122.4 (Ar-CH), 127.3 (Ar-CH), 133.3 (Ar-CH), 140.3 (Ar-C), 171.5 (CONH2
Or CONH), 172.8 (CONH2 Or CONH) ppm.
IR= 3432 (NH), 3351 (OH), 1663 (C=O), 1509, 1381, 1283, 1023 (R2CH-OH) cm-1
.
MS (+ESI) m/z= Found 495.3577 (M+H)+; calculated for C31H47N2O3 495.3581; 0.8 ppm.
- 101 -
Preparation of 2-[[(4R)-4-[(3R,10S,12S,13R,17R)-3,12-dihydroxy-10,13-dimethyl-
2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-
yl]pentanoyl]amino]benzamide (compound 85)
Deoxycholic acid (2.0 g 0.005 mol) was dissolved in 1, 4-dioxane (40 mL) with triethylamine (1.40
mL, 0.01 mol). The solution was cooled for 10 minutes in cold water. Ethyl chloroformate (0.54 mL,
0.005 mol) was then added dropwise over a ten minute period. Once added, the cold water was
removed and the solution was stirred for 2 hours. After 2 hours 2-aminobenzamide (0.7 g, 0.005 mol)
was added and the solution was stirred for a further 48 hours. Water (50 mL) was then added and the
solution was extracted with ethyl acetate (3 x 50 mL). The organic layers were combined and washed
with saturated sodium hydrogen carbonate solution (3 x 50 mL). The organic layer was dried over
magnesium sulfate. The solvent was evaporated under reduced pressure and the product was triturated
with diethyl ether and then vacuum filtered to produce a white powder. TLC EtOAc: MeOH 4:1 Rf
0.25 (single spot).
Yield; 1.78 g, 0.003 mol, 68 %.
Melting point: 119.8 – 125.7 oC.
1H NMR (d6 DMSO) (250 MHz) δ= 0.60 (s, 3H, 18-CH3), 0.85 (s, 3H, 19-CH3), 1.00-2.38 (m, 33H,
steroidal backbone CH/CH2), 3.80 (s, 1H, 12-CH), 4.22 (d, 1H, 3-OH, J= 5.0), 4.47 (d, 1H, 12-OH, J=
- 102 -
5.0), 7.10 (t, 1H, Ar-CH, J= 10.0), 7.48 (t, 1H, Ar-CH, J= 10.0), 7.72 (s, 1H, split NH2), 7.79 (d, 1H,
Ar-CH, J= 7.5), 8.25 (s, 1H, NH2), 8.47 (d, 1H, CH, J= 7.5), 11.71 (s, 1H, NH) ppm.
13C NMR (d6 DMSO) (62.9 MHz) δ= 12.4 (CH3, C18), 15.1 (CH2), 16.9 (CH3, C21), 23.0 (CH3, C19),
24.4 (CH2, C15), 26.0 (CH2, C7), 26.9 (CH2, C6), 27.1 (CH2, C16), 28.6 (CH2, C11), 30.2 (CH2, C2),
31.3 (CH2, C23), 32.9 (CH2, C22), 33.7 (CH, C9), 34.7 (C, C10), 34.9 (CH2, C1), 35.1 (CH, C20),
36.2 (CH2, C4), 38.4 (CH, C8), 45.9 (CH, C5), 46.1 (C, C13), 47.4 (CH, C17), 64.8 (CH), 69.9 (CH,
C3), 70.9 (CH2, C12), 119.2 (Ar-CH), 119.9 (Ar-CH), 122.0 (Ar-CH), 128.4 (Ar-C), 132.1 (Ar-C),
139.7 (Ar-CH), 170.7 (CONH2 Or CONH), 171.4 (CONH2 Or CONH) ppm.
IR= 3343 (OH), 2925 (alkyl), 2861 (alkyl), 1663 (C=O), 1189.78 (C=O ester stretch), 1518, 1441,
1381, 1270, 1023 (RCHOH), 750 cm-1
.
MS (ES) m/z= Found 511.3525 (M+H)+; calculated for C31H47N2O4 511.3530; 1.0 ppm.
Preparation of 2-[4-[(3R,7R,10S,12S,13R,17R)-3,7,12-trihydroxy-10,13-dimethyl-
2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-
yl]pentanoylamino]benzamide (compound 86)
Cholic acid (2.0 g 0.005 mol) was dissolved in 1, 4-dioxane (40 mL) with triethylamine (1.40 mL 0.01
mol). The solution was cooled for 10 minutes in cold water. Ethyl chloroformate (0.54 mL 0.005 mol)
was then added dropwise over a ten minute period. Once added, the cold water was removed and the
solution was stirred for 2 hours. After 2 hours 2-aminobenzamide (0.7 g 0.005 mol) was added and the
- 103 -
solution was stirred for a further 48 hours. Water (50 mL) was then added and the solution extracted
with ethyl acetate (3 x 50 mL). The organic layers were combined and washed with saturated sodium
hydrogen carbonate solution (3 x 50 mL). The organic layer was dried over magnesium sulfate.
Solvent was evaporated under reduced pressure and recrystallized from ethyl acetate to produce a
white powder. TLC EtOAc: MeOH 4:1 Rf 0.25 (single spot).
Yield: 0.692 g, 0.001 mol, 26.8 %.
Melting point: 122.8 – 129.9 oC.
1H NMR (d6 DMSO) (250 MHz) δ= 0.60 (s, 3H, 18-CH3), 0.82 (s, 3H, 19-CH3), 0.99 (d, 3H, 21-CH3,
J= 7.5), 1.35-2.50 (m, 33H, steroidal backbone CH/CH2), 3.63 (s, 1H, 3-CH), 3.80 (s, 1H, 12-CH),
4.00 (d, 1H, 7-OH), 4.14 (s, 1H, 3-OH), 4.34 (s, 1H, 12-OH), 7.10 (t, 1H, Ar-CH, J= 7.50), 7.51 (t,
1H, Ar-CH, J= 15), 7.79 (d, 1H, Ar-CH), 8.25 (s, 1H, NH), 8.49 (d, 1H, Ar-CH), 11.70 (s, 1H, NH)
ppm.
13C NMR (d6 DMSO) (62.9 MHz) δ= 12.3 (CH3, C18), 17.0 (CH3, C21), 22.6 (CH3, C19), 22.7 (CH2,
C15), 26.1 (CH, C9), 27.2 (CH2, C16), 28.5 (CH2, C11), 30.3 (CH2, C3), 31.3 (CH2, C23), 34.3 (CH2,
C22), 34.8 (C, C10), 35.0 (CH2, C1), 35.2 (CH, C20), 41.3 (CH2, C4), 41.4 (CH, C8), 45.7 (CH), 46.0
(CH), 66.2 (CH, C7), 70.4 (CH, C3), 70.9 (CH, C12), 119.3 (Ar-C), 119.98 (Ar-CH), 122.10 (Ar-CH),
128.5 (Ar-CH), 132.1 (Ar-CH), 139.7 (Ar-C), 170.8 (CONH2 Or CONH), 171.5 (CONH2 Or CONH)
ppm.
IR= 3462 (OH), 2933, 2870 (alkyl), 1671 (C=O), 1189, 1091 (R2CH-OH) cm-1
.
MS (ES) m/z= Found 527.3472 (M+H)+; calculated for C31H47N2O5 527.3479; 1.4 ppm.
- 104 -
Preparation of (4R)-N-(4-aminobutyl)-4-[(3R,10S,12S,13R,17R)-3,12-dihydroxy-10,13-dimethyl-
2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-
yl]pentanamide (compound 87)
Methyl deoxycholate (1.0 g 0.009 mol) was dissolved in methanol (10 mL) with 1,4-diaminobutane
(2.47 mL 0.02 mol) was added and the solution was stirred for a further 72 hours. Water (50 mL) was
then added and the resultant precipitate was collected by vacuum filtration, washed with water (3 x 20
mL) and dried under vacuum to produce a white powder. TLC EtOAc: MeOH 4:1 Rf 0.25 (single
spot).
Yield; 0.29 g, 0.0006 mol, 25 %.
Melting point: 137.4-141 oC.
1H NMR (d6 DMSO) (250 MHz) δ= 0.58 (s, 3H, 18-CH3), 0.84 (s, 3H, 19-CH3), 0.91 (d, 3H, 21-CH3,
J= 5.0), 1.00-2.23 (m, 33H, steroidal backbone CH/CH2), 2.99 (q, 2H, CH2, J=7.5), 3.6 (m, 1H, 3-CH),
3.78 (s, 1H, 12-CH), 7.73 (broad s, 1H, NH) ppm.
13C NMR (d6 DMSO) (62.9 MHz) δ= 12.4 (CH3, C18), 17.0 (CH3, C21), 23.0 (CH3, C19), 23.4 (CH2,
C15), 26.0, (CH2, NH-CH2 or CH2N(CH3)2 or CH2CH2CH2), 26.6 (CH2, CH2, NH-CH2 or CH2N(CH3)2
or CH2CH2CH2), 26.9 (CH2, CH2, NH-CH2 or CH2N(CH3)2 or CH2CH2CH2), 27.1 (CH2, C7), 28.5
(CH2, C16), 30.2 (CH2, C11), 30.5 (CH2, C2), 31.7 (CH2, C23), 32.5 (CH2, C22), 32.8 (CH, C9), 33.7
(C, C10), 35.0 (CH2, C1), 35.1 (CH, C20), 35.6 (CH2, C4), 36.2 (CH, C8), 41.3 (CH, C5), 41.5 (CH),
- 105 -
45.9 (C, C13), 46.1 (CH, C17), 47.4 (CH, C14), 69.8 (CH, C3), 69.8 (CH2, C12), 172.2 (CO, C24)
ppm.
IR= 3322 (OH), 2929 (alkyl), 2857 (alkyl), 1624 (C=O), 1539, 1437, 1360, 1040 (R2CH-OH) cm-1
.
MS (+ESI) m/z= Found 463.3886 (M+H)+; calculated for C28H51N2O3 463.3894; 1.8 ppm.
Preparation of (4R)-N-[2-[2-(2-aminoethoxy)ethoxy]ethyl]-4-[(3R,10S,13R,17R)-3-hydroxy-
10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-
17-yl]pentanamide (compound 88)
Lithocholic acid (2.0 g, 0.005 mol) was dissolved in Toluene (40 mL). 2, 2’-
(Ethylenedioxy)bis(ethylamine) (7.8 mL, 0.052 mol) was added and the solution was heated at reflux
for 5 days. Water (50 mL) was then added and resultant precipitate collected by vacuum filtration,
with further washes of water (3 x 20 mL). Dried under vacuum to produce an off-white powder. TLC
EtOAc: MeOH 4:1 Rf 0.25 (single spot).
Yield, 1.62 g, 0.003 mol, 60.22 %
Melting point: 81.1-85.5 oC.
1H NMR (CDCl3) (250 MHz) δ= 0.64 (s, 3H, 18-CH3), 0.92 (s, 3H, 19-CH3), 1.00-2.29 (m, 33H,
steroidal backbone CH/CH2), 2.91 (broad s, 2H, CH2), 3.46-3.63 (multiple overlapping multiplets,
10H, CH2), 6.24 (broad s, 1H, NH) ppm.
- 106 -
13C NMR (CDCl3) (62.9 MHz) δ= 12.0 (CH3, C18), 18.4 (CH3, C21), 19.9 (CH2), 20.8 (CH2, C11),
23.3 (CH3, C19), 24.2 (CH2, C15), 26.4 (CH2, C7), 27.2 (CH2, C6), 28.2 (CH2, C16), 30.5 (CH2, C2),
31.7 (CH2, C22), 33.4 (CH2, C23), 34.5 (C, C10), 35.3 (CH, C20), 35.5 (CH2, C1), 35.8 (CH, C8),
36.4 (CH2, C4), 39.0 (CH2, C12), 39.1 (CH, C9), 40.2 (CH2, NH-CH2 or OCH2), 40.4 (CH2, CH2, NH-
CH2 or OCH2), 42.1 (CH, C5), 42.7 (C, C13), 56.0 (CH, C17), 56.5 (CH, C14), 70.1 (CH2), 70.5 (CH,
C3), 71.7 (CH2), 173.7 (CO, C24) ppm.
IR= 3356 (OH), 2921 (alkyl), 2857 (alkyl), 1641 (C=O), 1552, 1441, 1300, 1104 (C=O ester stretch),
1053 (R2CH-OH) cm-1
.
MS (+ESI) m/z= Found 507.4149 (M+H)+; calculated for C30H55N2O4 507.4156; 1.4 ppm.
Preparation of (4R)-N-[2-[2-(2-aminoethoxy)ethoxy]ethyl]-4-[(3R,7R,10S,12S,13R,17R)-3,7,12-
trihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-
cyclopenta[a]phenanthren-17-yl]pentanamide (compound 89)
Cholic acid (2.0 g 0.005 mol) was dissolved in tetrahydrofuran (40 mL) with triethylamine (1.78 mL,
0.01 mol). The solution was cooled for 10 minutes in cold water. Ethyl chloroformate (0.52 mL, 0.005
mol) was then added dropwise over a ten minute period. Once added, the cold water was removed and
the solution was stirred for 2 hours. After 2 hours this material was added to a flask containing 2, 2’
(ethylenedioxy)bis(ethylamine) (7.25 mL 0.04 mol) drop by drop and the solution was stirred for 24
hours. Water (50 mL) was then added and the solution was extracted with ethyl acetate (3 x 50 mL).
- 107 -
The organic layers were combined and washed with saturated sodium hydrogen carbonate solution (3
x 50 mL). The organic layer was dried over magnesium sulfate. The solvent was evaporated under
reduced pressure to produce a white powder. TLC EtOAc: MeOH 4:1 Rf 0.25 (single spot).
Yield; 0.05 g, 0.00009 mol, 1.9 %.
Melting point: 134.0-136.0 oC.
1H NMR (CDCl3) (250 MHz) δ= 0.67 (s, 3H, 18-CH3), 0.88 (s, 3H, 19-CH3), 1.28-1.92 (m, 33H,
steroidal backbone CH/CH2), 2.63 (broad s, 1H, split NH2), 3.04 (broad s, 1H, split NH2), 3.44-3.62
(multiple overlapping multiplets, 12H, CH2), 3.83 (s, 1H, 3-CH), 3.96 (s, 1H, 12-CH), 6.73 (broad s,
1H, NH) ppm.
13C NMR (CDCl3) (62.9 MHz) δ= 12.4 (CH3, C18), 14.2 (CH2), 17.5 (CH3, C21), 21.0 (CH2), 22.4
(CH3, C19), 23.3 (CH2, C15), 26.3 (CH, C9), 27.6 (CH2, C16), 28.1 (CH2), 30.5 (CH2, C11), 31.8
(CH2, C3), 32.9 (CH2, C23), 34.8 (CH2, C6), 35.4 (C, C10), 39.1 (CH2, C1), 39.5 (CH, C20), 39.6
(CH2), 41.5 (CH2, C4), 46.3 (CH, C5), 46.4 (C, C13), 60.4 (CH2), 68.4 (CH, C7), 69.9 (CH2), 70.1
(CH2), 71.8 (CH, C3), 73.1 (CH, C12), 174.4 (C, CO) ppm.
IR= 3334 (OH), 2925 (alkyl), 2861 (alkyl), 1646 (C=O), 1535, 1449, 1368, 1066, 1040 (R2CH-OH)
cm-1
.
MS (+ESI) m/z= No corresponding peak found.
- 108 -
Preparation of (4R)-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-
2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]-N-[2-[2-[2-
(2-methylprop-2-enoylamino)ethoxy]ethoxy]ethyl]pentanamide (compound 90)
Using a modification of a method from (Hu et al., 2005) product from compound 88 (0.5 g 0.0009
mol) was dissolved in anhydrous tetrahydrofuran (10 mL) with triethylamine (0.20 mL, 0.001 mol).
Methacrylic anhydride (0.30 mL, 0.0018) was then added. The solution was stirred for 48 hours,
protected from sunlight by tin foil at ambient temperature. Water (30 mL) was then added and the
resulting precipitate was collected by vacuum filtration, washed with water (3 x 20 mL) and dried
under vacuum. TLC EtOAc: MeOH 4:1 Rf 0.25 (single spot).
Yield; 0.07 g, 0.0001 mol, 12.5 %.
Melting point: 57.3 – 59.0 oC.
1H NMR (d6 DMSO) (250 MHz) δ= 0.60 (s, 3H, 18-CH3), 0.87 (s, 3H, 19-CH3), 1.00-2.38 (m, 33H,
steroidal backbone CH/CH2), 1.84 (s, 3H, acryloyl-CH3), 3.154-3.50 (m, 13H, CH/CH2), 4.42 (d, 1H,
3-OH, J= 5.0), 5.31 (s, 1H, =CH), 5.64 (s, 1H, =CH), 7.78 (broad s, 1H, NH), 7.91 (broad s, 1H, NH)
ppm.
- 109 -
13C NMR (d6 DMSO) (62.9 MHz) δ= 11.8 (CH3, C18), 18.2 (CH3, C21), 18.6 (CH), 20.3 (CH2, C11),
23.2 (CH3, C19), 23.8 (CH2, C15), 26.1 (CH2, C7), 26.8 (CH2, C6), 27.7 (CH2, C16), 30.3 (CH2, C2),
31.5 (CH2, C22), 32.2 (CH2, C23), 34.1 (C, C10), 34.9 (CH, C20), 35.1 (CH2, C1), 35.3 (CH, C8),
41.4 (CH, C5), 42.2 (C, C13), 55.2 (CH, C17), 56.0 (CH, C14), 68.8 (CH2, CH2, NH-CH2 or OCH2),
69.1 (CH2, CH2, NH-CH2 or OCH2), 69.5 (CH2, CH2, NH-CH2 or OCH2), 69.8 (CH, C3), 118.9 (=CH2),
139.8 (=C), 167.4 (CO), 172.5 (CO, C24) ppm.
IR= 3415 (NH), 3292 (OH), 2933 (alkyl), 2861 (alkyl), 1658 (C=O), 1590 (C=O), 1394, 1249 (C-O
ester stretch), 1036 (R2CH-OH) cm-1
.
MS (+ESI) m/z= Found 575.4412 (M+H)+; calculated for C34H59N2O5 575.4418; 1.1 ppm.
Preparation of 2-[2-[2-[2-[[(4R)-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-
2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-
yl]pentanoyl]amino]ethoxy]ethoxy]ethylamino]benzamide (compound 91)
Compound 88 (0.5 g 0.0009 mol) was dissolved in ethanol (15 mL) and isatoic anhydride (0.17 g,
0.001 mol) was added. The solution was heated at reflux for 24 hours then allowed to cool. Water (50
mL) was then added and the resulting precipitate was collected by vacuum filtration and washed with
hot water (3 x 20 mL) to produce a white powder. TLC EtOAc: MeOH 4:1 Rf 0.25 (single spot).
Yield; 0.15 g, 0.0002 mol, 24.5 %.
Melting point: 72.5-74.7 oC.
- 110 -
1H NMR (CDCl3) (250 MHz) δ= 0.63 (s, 3H, 18-CH3), 0.91 (s, 3H, 19-CH3), 1.00-2.38 (m, 33H,
steroidal backbone CH/CH2), 3.43 (t, 2H, CH2, J= 7.5), 3.52 (t, 2H, CH2, J= 5.0), 3.63 (m, 8H, CH2),
5.96 (broad s, 1H, NH), 6.66 (overlapping multiplets, 2H, Ar-CH), 7.20, (t, 2H, Ar-CH, J= 5.0), 7.36
(d, 1H, Ar-CH J = 12.5 and 15) ppm.
13C NMR (CDCl3) (62.9 MHz) δ= 12.0 (CH3, C18), 18.3 (CH3, C21), 20.8 (CH2, C11), 23.3 (CH3,
C19), 24.2 (CH2, C15), 26.4 (CH2, C7), 27.2 (CH2, C6), 28.2 (CH2, C16), 30.5 (CH2, C2), 31.7 (CH2,
C22), 33.5 (CH2, C23), 34.5 (C, C10), 35.3 (CH, C20), 35.4 (CH2, C1), 35.8 (CH, C8), 36.4 (CH2,
C4), 39.1 (CH2, C12), 39.3 (CH, C9), 40.1 (CH2, CH2, NH-CH2 or OCH2), 40.4 (CH2, CH2, NH-CH2 or
OCH2) 42.1 (CH, C5), 42.7 (C, C13), 55.9 (CH, C17), 56.4 (CH, C14), 69.8 (CH2), 70.0 (CH2), 70.1
(CH2), 70.2 (CH2), 71.8 (CH, C3), 116.0 (Ar-CH), 116.5 (Ar-CH), 117.3 (Ar-CH), 127.3 (Ar-CH),
132.2 (Ar-CH), 148.7 (Ar-CH), 169.3 (CO), 173.7 (CO, C24) ppm.
IR= 3330 (OH), 2929 (alkyl), 2861 (alkyl), 1624 (C=O), 1535, 1432, 1258, 1087 (R2CH-OH) cm-1
.
MS (+ESI) m/z= Found 626.4523 (M+H)+; calculated for C37H60N3O5 626.4527; 0.7 ppm.
Preparation of (4R)-N-(4-acetylphenyl)-4-[(3R,10S,12S,13R,17R)-3,12-dihydroxy-10,13-
dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-
yl]pentanamide (compound 92)
Deoxycholic acid (2.0 g 0.005 mol) was dissolved in 1, 4 dioxane (60 mL) with triethylamine (1.32
mL, 0.01 mol). The solution was cooled for 10 minutes in cold water. Ethyl chloroformate (0.40 mL,
- 111 -
0.003 mol) was then added dropwise over a ten minute period. Once added, the cold water was
removed and the solution was stirred for 2 hours. After 2 hours 4’-aminoacetophenone (0.75 g, 0.005
mol) was added and the solution was stirred for a further 48 hours. Water (50 mL) was then added and
the resulting precipitate was collected by vacuum filtration. The crude product was triturated with
ethyl acetate to produce a white powder. TLC EtOAc: MeOH 4:1 Rf 0.25 (single spot).
Yield; 0.65 g, 0.001 mol, 25 %.
Melting point: Gradual softening from 194.1-206.9 oC.
1H NMR (d6 DMSO) (250 MHz) δ= 0.62 (s, 3H, 18-CH3), 0.86 (s, 3H, 19-CH3), 1.00-2.50 (m, 33H,
steroidal backbone CH/CH2), 2.52 (s, 3H, ketone-CH3), 3.81 (broad s, 1H, 12-CH), 4.21 (d, 1H, 3-OH,
J =2.5), 4.46 (d, 1H, 12-OH, J= 2.5), 7.73 (d, 2H, Ar-CH, J= 10.0), 7.92 (d, 2H, Ar-CH, J= 7.5), 10.21
(s, 1H, NH) ppm.
13C NMR (62.9 MHz) δ= Due to solubility issues, there was not a strong enough concentration of
material for a successful carbon NMR to be completed.
IR= 3475 (OH), 2921 (alkyl), 2861 (alkyl), 1684 (C=O), 1646 (C=O), 1594, 1539, 1407, 1044 cm-1
.
MS (+ESI) m/z= Found 510.3570 (M+H)+; calculated for C32H48NO4 510.3578; 1.3 ppm.
- 112 -
Preparation of (4R)-N-benzyl-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-
2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-
yl]pentanamide (compound 93)
Lithocholic acid (2.0 g 0.005 mol) was dissolved in tetrahydrofuran (60 mL) with N-
methylmorpholine (1.07 mL 0.01 mol). The solution was cooled for 10 minutes in cold water. Ethyl
chloroformate (0.51 mL 0.005 mol) was then added dropwise over a ten minute period. Once added,
the cold water was removed and the solution was stirred for 2 hours. After 2 hours 4-benzylamine (1.7
mL 0.01 mol) was added and the solution was stirred for a further 24 hours. Water (50 mL) was then
added and the solution extracted with ethyl acetate (3 x 50 mL). The organic layers were combined
and washed with saturated sodium hydrogen carbonate solution (3 x 50 mL). The crude product was
recrystallized from dichloromethane to produce a white powder.
Yield; 1.27 g, 0.002 mol, 51 %.
Melting point: 189.9-191.6 oC.
1H NMR (CDCl3) (250 MHz) δ= 0.64 (s, 3H, 18-CH3), 0.91 (d, 3H, 19-CH3, J= 2.5), 0.92 (s, 3H, 21-
CH3), 1.00-2.38 (m, 33H, steroidal backbone CH/CH2), 3.62 (m, 1H, 3-CH), 4.445 (d, 2H, CH2, J=
7.5), 7.32-7.34 (multiple overlapping aromatic multiplets, 5H, CH) ppm.
13C NMR (CDCl3) (62.9 MHz) δ= 12.0 (CH3, C18), 18.4 (CH3, C21), 20.8 (CH2, C11), 23.3 (CH3,
C19), 24.2 (CH2, C15), 26.4 (CH2, C7), 27.2 (CH2, C6), 28.2 (CH2, C16), 30.5 (CH2, C2), 31.8 (CH2,
C22), 33.6 (CH2, C23), 34.5 (C, C10), 35.3 (CH, C20), 35.4 (CH2, C1), 35.8 (CH, C8), 36.4 (CH2,
- 113 -
C4), 40.2 (CH2, C12), 40.4 (CH, C9), 42.1 (CH, C5), 42.7 (C, C13), 43.6 (CH2, CH2-Ar), 56.0 (CH,
C17), 56.5 (CH, C14), 71.8 (CH, C3), 127.5 (Ar-CH), 127.8 (Ar-CH), 128.7 (Ar-CH), 138.4 (Ar-C),
173.3 (CO, C24) ppm.
IR= 3398 NH), 3322 (OH), 2929 (alkyl), 2857 (alkyl), 1629 (C=O), 1552, 1445, 1364, 1228, 1044
(R2CH-OH) cm-1
.
MS (+ESI) m/z= Found 466.3672 (M+H)+; calculated for C31H48NO2 466.3680; 1.6 ppm.
Preparation of ethane; (4R)-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-
2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]-1-[4-(2-
hydroxyethyl)piperazin-1-yl]pentan-1-one (compound 94)
Lithocholic acid (4.0 g 0.01 mol) was dissolved in tetrahydrofuran (120 mL) with triethylamine (1.30
mL, 0.01 mol). The solution was cooled for 10 minutes in cold water. Ethyl chloroformate (1.02 mL,
0.009 mol) was then added dropwise over a ten minute period. Once added, the cold water was
removed and the solution was stirred for 2 hours. After 2 hours 1-(2-hydroxyethylpiperazine) (1.5 mL,
0.01 mol) was added and the solution was stirred for a further 48 hours. Water (50 mL) was then
added and the solution was extracted with ethyl acetate (3 x 50 mL). The organic layers were
combined and washed with saturated sodium hydrogen carbonate solution (3 x 50 mL). The organic
layer was dried over magnesium sulfate. The solvent was evaporated under reduced pressure and the
product was recrystallized from ethyl acetate to produce a white powder. TLC EtOAc: MeOH 4:1 Rf
0.25 (single spot).
- 114 -
Yield; 2.27 g, 0.004 mol, 43 %.
Melting point: 153.0-154.8 oC.
1H NMR (CDCl3) (250 MHz) δ= 0.65 (s, 3H, 18CH3), 0.92 (s, 3H, 19-CH3), 1.00-2.49 (m, 33H,
steroidal backbone CH/CH2), 2.50 (overlapping m, 4H, CH2), 2.58 (t, 2H, CH2, J= 5.0), 3.49 (t, 2H,
CH2, J= 2.5), 3.65 (t, 4H, CH2, J= 5.0) ppm.
13C NMR (CDCl3) (62.9 MHz) δ= 12.0 (CH3, C18), 18.5 (CH3, C21), 20.8 (CH2, C11), 23.3 (CH3,
C19), 24.2 (CH2, C15), 26.4 (CH2, C7), 27.2 (CH2, C6), 28.2 (CH2, C16), 30.2 (CH2, C2), 30.5 (CH2,
C22), 31.4 (CH2, C23), 34.5 (C, C10), 35.3 (CH, C20), 35.6 (CH2, C1), 35.8 (CH, C8), 36.4 (CH2,
C4), 40.2 (CH2, C12), 40.4 (CH, C9), 41.4 (CH, C5), 42.1 (C, C13), 42.7 (CH2), 45.6 (CH2), 52.6
(CH2), 53.1 (CH2), 56.0 (CH, C17), 56.5 (CH, C14), 57.7 (CH2), 59.3 (CH2), 71.8 (CH, C3), 172.1
(CO, C24) ppm.
IR= 3381 (OH), 2916 (alkyl), 2840 (alkyl), 1620 (C=O), 1445, 1258, 1044 (R2CH-OH) cm-1
.
MS (ES) m/z= Found 489.4045 (M+H)+; calculated for C30H53N2O3 489.4051; 1.2 ppm.
- 115 -
Preparation of (4R)-4-[(3R,10S,12S,13R,17R)-3,12-dihydroxy-10,13-dimethyl-
2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]-1-[4-(2-
hydroxyethyl)piperazin-1-yl]pentan-1-one (compound 95)
Deoxycholic acid (4.0 g 0.01 mol) was dissolved in 1, 4-dioxane (120 mL) with triethylamine (2.70
mL 0.02 mol). The solution was cooled for 10 minutes in cold water. Ethyl chloroformate (0.8 mL
0.005 mol) was then added dropwise over a ten minute period. Once added, the cold water was
removed and the solution was stirred for 2 hours. After 2 hours 1-(2-hydroxyethylpiperazine) (1.32
mL 0.01 mol) was added and the solution was stirred for 48 hours. Water (50 mL) was then added and
the solution extracted with ethyl acetate (3 x 50 mL). The organic layers were combined and washed
with saturated sodium hydrogen carbonate solution (3 x 50 mL). The organic layer was dried over
magnesium sulfate. Solvent was evaporated under reduced pressure and recrystallized from methanol
to produce a white powder. TLC EtOAc: MeOH 4:1 Rf 0.2 (single spot).
Yield; 2.41 g, 0.004 mol, 46.8 %.
Melting point: 241.0-243.9 oC.
1H NMR (CDCl3/MeOH) (250 MHz) δ= 0.70 (s, 3H, 18-CH3), 0.93 (s, 3H, 19-CH3), 1.04-2.50 (m,
33H, steroidal backbone CH/CH2), 2.58 (multiple overlapping multiplets, 6H, CH2), 3.53 (broad t, 3H,
3-CH/CH2), 3.65-3.72 (multiple overlapping multiplets, 4 H, CH2), 3.96 (broad s, 1H, 12-CH) ppm.
- 116 -
13C NMR (d6-DMSO) (250 MHz) δ= 12.4 (CH3, C18), 17.0 (CH3, C21), 23.0 (CH3, C19),
28.5 (CH2, C16), 29.4 (CH2, C11), 30.1 (CH2, C2), 32.8 (CH2, C23), 33.7 (CH2, C22), 35.12
(CH, C9), 35.56 (C, C10), 36.22 (CH2, C1), 45.9 (C, C13), 46.1 (CH, C17), 47.4 (CH, C14),
52.89 (CH2), 53.43 (CH2), 58.38 (CH2), 69.9 (CH, C3), 71.0 (CH2, C12), 78.33 (CH2), 78.8
(CH2), 79.0 (CH2), 79.3 (CH2), 171.0 (CO, C24) ppm.
IR= 3407 (OH), 2929 (alkyl), 1620 (C=O), 1454, 1215, 1044 (R2CH-OH) cm-1
.
MS (+ESI) m/z= Found 505.3994 (M+H)+; calculated for C31H57N2O4 505.4000; 1.2 ppm.
Preparation of (4R)-N-[3-(dibutylamino)propyl]-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-
dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-
yl]pentanamide (compound 96)
Lithocholic acid (0.5 g 0.001 mol) was dissolved in tetrahydrofuran (15 mL) with triethylamine (0.20
mL 0.001 mol). The solution was cooled for 10 minutes in cold water. Ethyl chloroformate (0.2 mL
0.001 mol) was then added dropwise over a ten minute period. Once added, the cold water was
removed and the solution was stirred for 2 hours. After 2 hours 3-(dibutylamino)-1-propylamine (0.25
mL, 0.001 mol) was added and the solution was stirred for 24 hours. Water (50 mL) was then added
and the solution extracted with ethyl acetate (3 x 50 mL). The organic layers were combined and
washed with saturated sodium hydrogen carbonate solution (3 x 50 mL). This crude material was
- 117 -
further purified using column chromatography (8:2 ethyl acetate/methanol). The compound containing
fractions were dried over magnesium sulfate. Solvent was evaporated under reduced pressure to
produce a white powder.
Yield; 0.24 g, 0.0004 mol, 33%.
Melting point: 62.9 – 69.3 oC.
1H NMR (CDCl3) (250 MHz) δ= 0.64 (s, 3H, 18-CH3), 0.93 (s, 3H, 19-CH3), 0.94 (m, 6H, CH3), 1.00-
2.38 (m, 33H, steroidal backbone CH/CH2), 1.35 (m, 8H, CH2), 2.45 (t, 4H, CH2, J= 5.0), 3.32 (q, 2H,
CH2, J=5.0), 7.51 (broad s, 1H, NH) ppm.
13C NMR (CDCl3) (62.9 MHz) δ= 12.0 (CH3, C18), 14.0 (CH3 CH2-CH3), 18.3 (CH3, C21), 20.7 (CH2,
C11), 23.3 (CH3, C19), 24.2 (CH2, C15), 25.1 (CH2), 26.4 (CH2, C7), 27.2 (CH2, C6), 28.2 (CH2,
C16), 28.4 (CH2), 28.5 (CH2), 30.4 (CH2, C2), 30.5 (CH2, C22), 33.8 (CH2, C23), 34.5 (C, C10), 35.3
(CH, C20), 36.4 (CH2, C1), 39.4 (CH2, C4), 40.1 (CH2, C12), 40.4 (CH, C9), 42.0 (CH, C5), 42.1 (C,
C13), 42.7 (CH2), 56.1 (CH, C17), 56.4 (CH, C14), 71.6 (CH, C3), 173.6 (CO, C24) ppm.
IR= 3296 (OH), 2916 (alkyl), 2861 (alkyl), 1650 (C=O), 1548, 1441, 1377, 1070, 1066, 1036 (R2CH-
OH) cm-1
.
MS (+ESI) m/z= Found 545.5032 (M+H)+; calculated for C35H65N2O2 545.5041; 1.6 ppm.
- 118 -
Preparation of (4R)-N-[3-(dibutylamino)propyl]-4-[(3R,10S,12S,13R,17R)-3,12-dihydroxy-10,13-
dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-
yl]pentanamide (compound 97)
Deoxycholic acid (0.5 g 0.001 mol) was dissolved in tetrahydrofuran (15 mL) with triethylamine (0.20
mL 0.001 mol). The solution was cooled for 10 minutes in cold water. Ethyl chloroformate (0.2 mL
0.001 mol) was then added dropwise over a ten minute period. Once added, the cold water was
removed and the solution was stirred for 2 hours. After 2 hours 3-(dibutylamino)-1-propylamine (0.25
mL 0.001 mol) was added and the solution was stirred for a further 48 hours. Water (50 mL) was then
added and the solution extracted with ethyl acetate (3 x 50 mL). The organic layers were combined
and washed with saturated sodium hydrogen carbonate solution (3 x 50 mL). This crude material was
further purified using column chromatography (8:2 ethyl acetate/methanol). The compound containing
fractions were dried over magnesium sulfate. Solvent was evaporated under reduced pressure to
produce viscous oil.
Yield; 0.03g, 0.00005 mol, 4 %.
Melting point: Oil.
- 119 -
1H NMR (CDCl3) (250 MHz) δ= 0.67 (s, 3H, 18-CH3), 0.91 (s, 3H, 19-CH3), 0.91 (m, 6H, CH3), 1.30-
2.22 (m, 33H, steroidal backbone CH/CH2), 1.48 (m, 8H, CH2), 2.50 (t, 4H, CH2, J= 5.0), 3.32 (q, 2H,
CH2, J=5.0), 3.58 (m, 1H, CH), 4.08 (s, 1H, CH), 7.51 (broad s, 1H, NH) ppm.
13C NMR (CDCl3) (62.9 MHz) δ= 12.7 (CH3, C18), 14.0 (CH3), 17.5 (CH3, C21), 20.6 (CH2), 23.1
(CH3, C19), 23.7 (CH2, C15), 25.0 (CH2), 26.1 (CH2), 27.1 (CH2, C7), 27.5 (CH2), 28.5 (CH2, C6),
28.8 (CH2, C16), 30.4 (CH2, C11), 31.6 (CH2, C2), 33.3 (CH2, C23), 34.1 (CH, C9), 35.3 (C, C10),
36.0 (CH2, C1), 36.4 (CH, C20), 43.1 (CH, C5), 46.5 (C, C13), 46.9 (CH, C17), 48.2 (CH, C14), 53.2
(CH2), 53.8 (CH2), 71.7 (CH, C3), 73.1 (CH2, C12), 173.7 (CO, C24) ppm.
IR= 3309 (OH), 2925 (alkyl), 2857 (alkyl), 1701 (C=O), 1650, 1445, 1249 (C=O ester stretch), 1044
(R2CH-OH) cm-1
.
MS (+ESI) m/z= Found 561.4982 (M+H)+; calculated for C35H65N2O3 561.4990; 1.4 ppm.
Preparation of (4R)-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-
2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]-N-
octadecyl-pentanamide (compound 98)
Lithocholic acid (2.0 g 0.005 mol) was dissolved in tetrahydrofuran (60 mL) with 4-methylmorpholine
(2.14 mL, 0.02 mol). The solution was cooled for 10 minutes in cold water. Ethyl chloroformate (0.51
mL, 0.005 mol) was then added dropwise over a ten minute period. Once added, the cold water was
- 120 -
removed and the solution was stirred for 2 hours. After 2 hours octadecylamine (1.43 mL, 0.005 mol)
was added and the solution was stirred for a further 24 hours. Water (50 mL) was then added and the
solution was extracted with ethyl acetate (3 x 50 mL). The crude product was purified by column
chromatography (100 % ethyl acetate). The product containing fractions were dried over magnesium
sulfate. The solvent was evaporated under reduced pressure to produce a white powder.
Yield; 0.8 g, 0.001 mol, 23.9 %.
Melting point: 94.8-95.4 oC.
1H NMR (250 MHz) CDCl3 δ= 0.64 (s, 3H, 18-CH3), 0.92 (s, 3H, 19-CH3), 1.00-2.38 (m, 33H,
steroidal backbone CH/CH2), 1.25 (s, 30H, aliphatic CH2), 3.23 (q, 3H, chain terminal CH3, J=5.0),
3.64 (m, 1H, 3-CH), 5.36 (broad s, 1H, NH) ppm.
13C NMR (CDCl3) (62.9 MHz) δ= 12.0 (CH3, C18), 18.4 (CH3, C21), 20.8 (CH2, C11), 22.7 (CH3,
C19), 23.3 (CH2, C15), 24.2 (CH2), 26.4 (CH2, C7), 26.9 (CH2, C6), 27.2 (CH2, C16), 28.2 (CH2,
aliphatic side chain), 29.3 (CH2, aliphatic side chain), 29.5 (CH2, aliphatic side chain), 29.7 (CH2,
aliphatic side chain), 30.5 (CH2, C2), 31.8 (CH2, C22), 31.9 (CH2, C23), 33.7 (C, C10), 35.3 (CH,
C20), 35.4 (CH2, C1), 35.8 (CH, C8), 39.5 (CH2, C4), 40.2 (CH2, C12), 40.4 (CH, C9), 42.1 (C, C5),
56.0 (CH, C17), 56.5 (CH, C14), 71.9 (CH, C3), 185.3 (CO, C24) ppm.
IR= 3411 (NH), 3325 (OH), 2912 (alkyl), 2849 (alkyl), 1653 (C=O), 1544, 1464, 1367, 1308, 1040
(R2CH-OH) cm-1
.
MS (+ESI) m/z= Found 628.6205 (M+H)+; calculated for C42H78NO2 628.6207; 0.6 ppm.
- 121 -
Preparation of (4R)-4-[(3R,10S,12S,13R,17R)-3,12-dihydroxy-10,13-dimethyl-
2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]-N-
octadecyl-pentanamide (compound 99)
Deoxycholic acid (2.0 g 0.005 mol) was dissolved in 1, 4 dioxane (60 mL) with triethylamine (1.32
mL, 0.01 mol). The solution was cooled for 10 minutes in cold water. Ethyl chloroformate (0.40 mL,
0.003 mol) was then added dropwise over a ten minute period. Once added, the cold water was
removed and the solution was stirred for 2 hours. After 2 hours octadecylamine (1.37 g, 0.005 mol)
was added and the solution was stirred for a further 48 hours. Water (50 mL) was then added and the
solution was extracted with ethyl acetate (3 x 50 mL). The organic layers were combined and washed
with saturated sodium hydrogen carbonate solution (3 x 50 mL). The organic layer was dried over
magnesium sulfate. The solvent was evaporated under reduced pressure and the product was
recrystallized from ethyl acetate to produce a transparent glass like solid.
Yield; 2.32 g, 0.003 mol, 70 %.
Melting point: 54.2 – 57.3 oC.
- 122 -
1H NMR (CDCl3) (250 MHz) δ= 0.68 (s, 3H, 18-CH3), 0.91 (s, 3H, 19-CH3), 1.00-2.38 (m, 33H,
steroidal backbone CH/CH2), 1.25 (s, 32H, aliphatic CH2), 3.22 (q, 2H, CH2, J= 7.5), 3.61 (m, 1H, 3-
CH), 3.98 (1H, 12-CH), 5.53 (broad s, 1H, NH) ppm.
13C NMR (62.9 MHz) δ= 12.7 (CH3, C18), 14.1 (CH2), 17.4 (CH3, C21), 22.6 (CH2), 23.1 (CH3, C19),
23.6 (CH2, C15), 26.1 (CH), 26.9 (CH2, C7), 28.6 (CH2, C16), 29.3 (CH2, C11), 29.5 (CH2, C2), 29.7
(CH2), 30.5 (CH), 31.7 (CH2, C23), 31.9 (CH2, C22), 33.5 (CH, C9), 33.6 (CH), 34.1 (CH), 35.2 (C,
C10), 35.2 (CH), 36.0 (CH2, C1), 36.4 (CH, C20), 39.5 (CH2, C4), 42.1 (CH, C5), 46.5 (C, C13), 47.2
(CH, C17), 48.2 (CH, C14), 71.7 (CH, C3), 73.1 (CH2, C12), 173.4 (CO, C24) ppm.
IR= 3292 (OH), 2912, 2852 (alkyl), 1637 (C=O), 1548, 1189, 1036 (RCHOH) cm-1
.
MS (+ESI) m/z= Found 644.5971 (M+H)+; calculated for C42H78NO3 644.5976; 0.8 ppm.
2.3.2 Manipulations of 3-OH on bile acid derivatives.
Preparation of methyl 4-[(3R,10S,13R,17R)-10,13-dimethyl-3-prop-2-enoyloxy-
2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoate
(compound 100)
Methyl lithocholate (0.25 g 0.0006 mol) was dissolved in chloroform (15 mL) with triethylamine (0.12
mL 0.001 mol). Acryloyl chloride (0.1 mL 0.0009 mol) was added and the solution was stirred for 24
- 123 -
hours. Water (50 mL) was then added and the resultant precipitate was collected by vacuum filtration,
washed with water (3 x 20 mL) and dried under vacuum.
Yield; 0.16 g, 0.0003 mol, 57 %.
Melting point: 152.5 – 156.7 oC.
1H NMR (CDCl3) (250 MHz) δ= 0.58 (s, 3H, 18-CH3), 0.87 (s, 3H, 19-CH3), 1.00-2.28 (m, 33H,
steroidal backbone CH/CH2), 3.59 (S, 3H, O-CH3), 4.73 (m, 1H, 3-CH), 5.74 (dd, 1H, =CH- J =
machine not sensitive to distinguish peaks), 6.01 (dd, 1H, =CH J= machine not sensitive enough to
distinguish peaks), 6.28 (dd, 1H, =CH, J = 2.5) ppm.
13C NMR (CDCl3) (62.9 MHz) δ= 11.0 (CH3, C18), 17.2 (CH3, C21), 19.8 (CH2, C11), 22.3 (CH3,
C19), 23.1 (CH2, C15), 23.4 (CH2, C7), 25.3 (CH2, C6), 26.0 (CH2, C16), 27.1 (CH2, C2), 30.0 (CH2,
C22), 32.0 (CH2, C23), 34.0 (C, C10), 34.3 (CH, C20), 39.1 (CH2, C12), 40.8 (CH, C9), 42.0 (CH,
C5), 54.9 (CH, C17), 55.4 (CH, C14), 73.5 (CH, C3), 128.0 (CH2 =CH-), 129.1 (=CH2), 164.7 (CO),
178.1 (CO, C24) ppm.
IR= 2925 (alkyl), 2857 (alkyl), 1735 (C=O), 1714 (C=O), 1428, 1394, 1206, 980, 805 (CH out of
plane bending) cm-1
.
MS (ES) m/z= Found 462.3574 (M+NH4)+; calculated for C28H48NO4 462.3578; 0.8 ppm.
- 124 -
Preparation of poly[(3R,10S,13R,17R)-17-[(1R)-4-(benzylamino)-1-methyl-4-oxo-butyl]-10,13-
dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl]
prop-2-enoate (compound 101)
Compound 100 (0.16 g, 0.0003 mol) was dissolved in ethanol (10 mL) with azobisisobutyronitrile
(0.005mg). The mixture was heated at reflux for 48 hours. After 48 hours the material was added to
water (30 mL) and the resulting precipitate was collected by vacuum filtration, washed with water (3 x
20 mL) and dried under vacuum to produce a white powder.
Yield; 0.027 g, 0.0000625 mol, 16.9 %.
1H NMR (CDCl3) (250 MHz) δ= 0.64 (s, 3H, 18-CH3), 0.93 (s, 3H, 19-CH3), 1.10-2.52 (m, 33H,
steroidal backbone CH/CH2), 3.66 (s, 3H, O-CH3), 4.72 (m, 1H, 3-CH) ppm.
13C NMR (CDCl3) (62.9 MHz) δ= 12.0 (CH3, C18), 18.2 (CH3, C21), 20.8 (CH2, C11), 23.3 (CH3,
C19), 24.1 (CH2, C15), 26.3 (CH2, C7), 27.0 (CH2, C6), 28.2 (CH2, C16), 31.0 (CH2, C22), 31.0 (CH2,
C23), 34.6 (C, C10), 35.3 (CH, C20), 35.8 (CH2), 40.1 (CH2, C12), 40.4 (CH, C9), 41.9 (CH, C5),
42.7 (C, C13), 56.0 (CH2), 56.4 (CH), 56.0 (CH, C17), 56.4 (CH, C14), 174.7 (CO, C24) ppm.
IR= 3423 (NH), 3326 (OH), 2932 (alkyl), 2862 (alkyl), 1724 (C=O), 1448, 1166 (C=O ester stretch),
1020 (R2CH-OH) cm-1
.
- 125 -
Preparation of [(3R,10S,13R,17R)-17-[(1R)-4-(benzylamino)-1-methyl-4-oxo-butyl]-10,13-
dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl]
prop-2-enoate (compound 102)
Product from Compound 93 (0.25 g 0.0005 mol) was dissolved in chloroform (15 mL) with
triethylamine (0.21 mL 0.002 mol). Acryloyl chloride (0.24 mL 0.002 mol) was added and the solution
was stirred for 5 days. Water (50 mL) was then added and the resulting precipitate was collected by
vacuum filtration, washed with water (3 x 20 mL) and dried under vacuum.
Yield: 0.023 g, 0.00004 mol, 6.8 %.
Melting point: 123.3 – 127.7 oC.
1H NMR (CDCl3) (250 MHz) δ= 0.64 (s, 3H, 18-CH3), 0.94 (s, 3H, 19-CH3), 1.10-2.38 (m, 33H,
steroidal backbone CH/CH2), 4.44 (d, 2H, CH2 J= 5.0), 4.80 (m, 1H, 3-CH), 5.67 (broad s, 1H, NH),
5.795, (dd, 1H, =CH- J= 10 and 2.5), 6.095 (dd, 1H, =CH J= 10.0 and 17.5), 6.35 (dd, 1H, =CH J= 2.5
and 17.5), 7.29-7.31 (overlapping multiplets, 5H, Aromatics) ppm.
13C NMR (CDCl3) (62.9 MHz) δ= 12.0 (CH3, C18), 18.4 (CH3, C21), 20.8 (CH2, C11), 23.3 (CH3,
C19), 24.2 (CH2, C15), 26.3 (CH2, C7), 26.6 (CH2, C6), 27.0 (CH2, C16), 28.2 (CH2, C2), 31.8 (CH2,
C22), 32.2 (CH2, C23), 33.6 (C, C10), 34.6 (CH, C20), 34.6 (CH2, C1), 35.0 (CH, C8), 35.5 (CH2,
C4), 35.8 (CH2, C12), 40.1 (CH, C9), 40.4 (CH, C5), 41.9 (C, C13), 42.7 (CH), 43.6 (CH =CH), 56.0
(CH, C17), 56.5 (CH, C14), 74.5 (CH, C3), 127.5 (Ar-CH), 127.8 (Ar-CH), 128.7 (Ar-CH), 129.1
(=CH-), 130.1 (=CH2), 138.4, (Ar-C), 165.7 (CO), 173.2 (CO, C24) ppm.
- 126 -
IR= 3302 (OH), 2956 (alkyl), 2932 (alkyl), 2862 (alkyl), 1712 (C=O), 1642 (C=O), 1278, 1017
(R2CH-OH) cm-1
.
MS (ES) m/z= Found 520.3782 (M+NH4) +
; calculated for C34H50NO3 520.3785; 0.6 ppm.
2.3.3 Quaternization of tertiary amines in bile amide derivatives.
Preparation of 2-[[(4R)-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-
2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-
yl]pentanoyl]amino]ethyl-trimethyl-ammonium iodide (compound 103)
Compound 62 (0.2 g, 0.0004 mol) was dissolved in chloroform (10 mL) with Iodomethane (0.31 mL
0.002 mol). The solution was left overnight at which point a precipitate had formed which was
collected by vacuum filtration, washed with water (3 x 20 mL) and dried under vacuum to produce a
white powder. TLC 100% MeOH Rf 0.1 (single spot).
Yield; 0.212 g, 0.0003 mol, 81.5 %.
Melting point: 212.8 – 216.1 oC.
1H NMR (MeOD) (250 MHz) δ= 0.69, (s, 3H, 18-CH3), 0.94 (s, 3H, 19-CH3), 1.00-2.35 (m, 33H,
steroidal backbone CH/CH2), 3.19, (s, 9H, 3 x CH3), 3.45 (t, 2H, al-CH2, J= 7.5), 3.55 (m, 1H, 3-CH),
3.64 (t, 2H, CH2-NR3 , J= 7.5) ppm.
- 127 -
13C NMR (MeOD) (62.9 MHz) δ= 11.0 (CH3, N-CH3), 12.5 (CH3, C18), 17.3 (CH3, C21), 18.8 (CH2),
21.9 (CH), 23.9 (CH2), 27.6 (CH), 28.3 (CH), 31.2 (CH), 33.0 (CH), 33.9 (CH), 36.9 (CH2), 37.2
(CH2), 41.9 (CH), 43.5 (C, C13), 57.4 (CH, C17), 57.9 (CH, C14), 65.8 (CH2), 72.4 (CH), 181.2 (CO)
ppm.
IR= 3368 (NH), 3245 (OH), 2938 (alkyl), 2852 (alkyl) 1639 (C=O), 1560, 1441, 1258, 1040 (R2CH-
OH) cm-1
.
MS (+ESI) m/z= Found 461.4105 (M-I)+; calculated for C29H53N2O2 461.4102; 0.7 ppm.
Preparation of 2-[[(4R)-4-[(3R,10S,12S,13R,17R)-3,12-dihydroxy-10,13-dimethyl-
2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-
yl]pentanoyl]amino]ethyl-trimethyl-ammonium iodide (compound 104)
Compound 63 (0.15 g 0.0003 mol) was dissolved in chloroform (5 mL). Iodomethane (0.22 mL, 0.001
mol) was added and the solution was stirred overnight at ambient temperature. The resultant
precipitate was collected via vacuum filtration and dried under vacuum overnight. Product was an off-
white solid. TLC 100% MeOH, Rf 0.1 (single spot).
Yield 0.09g, 0.0001 mol, 78.9 %.
Melting point: 160.1 – 169.1 oC.
- 128 -
1H NMR (MeOD) (250 MHz) δ= 0.70 (s, 3H, 18-CH3), 0.93 (s, 3H, 19-CH3), 1.02 (d, 3H, 21-CH3, J=
5.0), 1.04-2.38 (m, 33H, steroidal backbone CH/CH2), 3.20 (s, 9H, 3 x CH3), 3.47 (t, 2H, CH2, J= 7.5),
3.64 (t, 2H, CH2, J= 5.0), 3.95 (s, 1H, 12-CH) ppm.
13C NMR (MeOD) (62.9 MHz) δ= 13.2 (CH3, C18), 17.6 (CH3, C21), 23.7 (CH3, C19), 24.8 (CH2,
C15), 27.4 (CH2, C7), 28.4 (CH2, C6), 28.7 (CH2, C16), 29.9 (CH2, C11), 31.0 (CH2, C2), 33.0 (CH2,
C23), 33.8 (CH2), 34.6 (CH2), 36.4 (CH2, C1), 36.8 (CH, C20), 37.2 (CH2, C4), 37.4 (CH, C8), 43.6
(CH, C5), 54.0 (CH), 54.0 (CH), 54.1 (CH), 72.5 (CH, C3), 74.0 (CH2, C12), 177.3 (CO, C24) ppm.
IR= 3377 (NH), 3249 (OH), 2921 (alkyl), 2861 (alkyl), 1641 (C=O), 1573, 1454, 1373, 1249, 1031
(R2CH-OH) cm-1
.
MS (+ESI) m/z= Found 477.4060 (M-I)+; calculated for C29H53N2O3 477.4051; 1.9 ppm.
Preparation of ethyl-[3-[[(4R)-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-
2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-
yl]pentanoyl]amino]propyl]-dimethyl-ammonium iodide (compound 105)
Compound 62 (0.2 g 0.0004 mol) was dissolved in chloroform (10 mL). Ethyl Iodide (0.34 mL, 0.001
mol) was added and the solution was stirred overnight at ambient temperature. The resulting
precipitate was collected by vacuum filtration and was dried overnight under vacuum. The product
was an off-white solid. TLC 100% MeOH, Rf 0.1 (single spot).
- 129 -
Yield; 0.2 g, 0.0003 mol, 76 %.
Melting point: 120.0-123.0 oC.
1H NMR (MeOD) (250 MHz) δ= 0.69 (s, 3H, 18-CH3), 0.95 (s, 3H, 19-CH3), 1.00-2.38 (m, 33H,
steroidal backbone CH/CH2), 3.13 (s, 6H, 2 x CH3), 3.43 (m, 4H, 3-CH/CH2), 3.60 (q, 2H, CH2, J=
7.5) ppm.
13C NMR (MeOD) (62.9 MHz) δ= 8.4 (CH), 12.5 (CH3, C18), 18.8 (CH3, C21), 21.4 (CH2, C11), 21.9
(CH3, C19), 23.9 (CH2, C15), 25.2 (CH2, C7), 28.3 (CH2, C16), 29.3 (CH2, C2), 31.2 (CH2, C23), 33.0
(CH2), 33.9 (CH2), 34.2 (C, C10), 35.6 (CH, C20), 36.4 (CH2, C1), 37.1 (CH, C8), 37.2 (CH2, C4),
41.5 (CH, C9), 41.9 (CH, C5), 43.5 (C, C13), 43.9 (CH2), 51.1 (CH2), 57.3 (CH, C17), 57.9 (CH,
C14), 62.4 (CH, C3), 177.3 (CO, C24) ppm.
IR= 3373 (OH), 2942 (alkyl), 2844 (alkyl), 1646 (C=O), 1441, 1270, 1036 (R2CH-OH) cm-1
.
MS (+ESI) m/z= Found 475.4247 (M-I)+; calculated for C30H55N2O2 475.4258; 2.3 ppm.
Preparation of 3-[[(4R)-4-[(3R,10S,12S,13R,17R)-3,12-dihydroxy-10,13-dimethyl-
2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-
yl]pentanoyl]amino]propyl-ethyl-dimethyl-ammonium iodide (compound 106)
Compound 63 (0.2 g 0.0004 mol) was dissolved in chloroform (5 mL). Ethyl Iodide (0.62 mL, 0.003
mol) was added and the solution was stirred overnight at ambient temperature. The resulting
- 130 -
precipitate was collected by vacuum filtration and was dried overnight under vacuum. The product
was an off-white solid. TLC 100% MeOH, Rf 0.1 (single spot).
Yield; 0.13 g, 0.0002 mol, 52 %.
Melting point: 115.3 – 120.3 oC.
1H NMR (CDCl3) (250 MHz) δ= 0.70 (s, 3H, 18-CH3), 0.93 (s, 3H, 19-CH3), 1.02 (d, 2H, CH2), 1.03-
2.38 (m, 33H, steroidal backbone CH/CH2), 2.21 (m, 6H, 3-CH/CH2), 3.4 (broad s, 2H, CH2), 3.45-
3.61 (multiple overlapping multiplets, 8H, CH2) ppm.
13C NMR (MeOD) (62.9 MHz) δ= 8.4 (CH2), 12.4 (CH3, C18), 16.9 (CH3, C21), 21.9 (CH2), 22.9
(CH3, C19), 24.0 (CH2, C15), 26.6 (CH2, C7), 27.9 (CH2, C16), 29.1 (CH2, C11), 30.3 (CH2, C2), 32.2
(CH2, C23), 33.1 (CH2, C22), 34.0 (CH, C9), 34.5 (C, C10), 35.6 (CH2, C1), 36.0 (CH, C20), 36.4
(CH2, C4), 36.6 (CH, C8), 42.8 (CH, C5), 55.6 (CH), 57.1 (CH), 63.2 (CH), 71.7 (CH, C3), 73.2 (CH2,
C12), 176.6 (CO, C24) ppm.
IR= 3368 (NH), 3253 (OH), 2921 (alkyl), 2852 (alkyl), 1650 (C=O), 1522, 1445, 1364, 1253, 1036
(R2CH-OH) cm-1
.
MS (+ESI) m/z= Found 517.4354 (M-I)+; calculated for C32H57N2O3 517.4364; 1.9 ppm.
- 131 -
Preparation of allyl-[2-[[(4R)-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-
2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-
yl]pentanoyl]amino]ethyl]-dimethyl-ammonium bromide (compound 107)
Compound 62 (0.2 g, 0.0004 mol) was dissolved in a solution of chloroform (10 mL). Allyl bromide
(0.27 mL, 0.002 mol) was added and the solution was stirred overnight at which point a precipitate
was formed. The precipitate was collected by vacuum filtration and was dried overnight under
vacuum. The product was an off-white solid. TLC 100% MeOH, Rf 0.1 (single spot).
Yield; 0.13 g, 0.0002 mol, 54 %.
Melting point: 198.7 – 203.8 oC.
1H NMR (MeOD) (250 MHz) δ= 0.68 (s, 3H, 18-CH3), 0.94 (s, 3H, 19-CH3), 1.04-2.38 (m, 33H,
steroidal backbone CH/CH2), 3.12 (s, 6H, 2 x CH3), 3.39 (t, 2H, CH2), 3.65 (t, 2H, CH2), 5.70 (m, 2H,
=CH2), 6.10 (m, 1H, =CH-) ppm.
13C NMR (MeOD) (62.9 MHz) δ= 12.5 (CH3, C18), 18.8 (CH3, C21), 21.9 (CH2, C11), 23.9 (CH3,
C19), 25.2 (CH2, C15), 27.6 (CH2, C7), 28.3 (CH2), 28.0 (CH2, C16), 33.0 (CH2), 34.2 (C, C10), 37.1
(CH2), 41.5 (CH2), 41.9 (CH2), 43.8 (CH), 57.3 (CH2), 57.9 (CH2), 63.0 (CH2), 72.4 (CH, C3), 126.0
(=CH2), 129.8 (=CH-), 177.3 (CO, C24) ppm.
IR= 3266 (OH), 2929 (alkyl), 2848 (alkyl), 1646 (C=O), 1569, 1420, 1066, 1036 (R2CH-OH) cm-1
.
- 132 -
MS (+ESI) m/z= Found 487.4252 (M-Br)+; calculated for C31H55N2O2 487.4258; 1.2 ppm.
Preparation of allyl-[2-[[(4R)-4-[(3R,10S,12S,13R,17R)-3,12-dihydroxy-10,13-dimethyl-
2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-
yl]pentanoyl]amino]ethyl]-dimethyl-ammonium iodide (compound 108)
Compound 63 (0.15 g, 0.0003 mol) was dissolved in chloroform (5 mL). Allyl bromide (0.20 mL,
0.0016 mol) was added and the solution was stirred overnight at ambient temperature. The resulting
precipitate was collected by vacuum filtration and was dried overnight under vacuum. The product
was an off-white solid. TLC 100% MeOH, Rf 0.1 (single spot).
Yield; 0.19 g, 0.0001 mol, 53 %.
Melting point: 184.2 – 186.7 oC.
1H NMR (MeOD) (250 MHz) δ= 0.70 (s, 3H, CH3), 0.93 (s, 3H, CH3), 1.12-2.30 (m, 33H, steroidal
backbone CH/CH2), 3.13 (s, 6H, CH3), 3.40 (t, 2H, CH2, J= 7.5), 3.65 (t, 2H, CH2, J= 5.0), 3.95 (s, 1H,
12-CH), 4.05 (d, 2H, CH2, J= 5.0), 5.75 (m, 2H, CH2), 6.10 (m, 1H, =CH-) ppm.
13C NMR (MeOD) (62.9 MHz) δ= 13.2 (CH3, C18), 17.6 (CH3, C21), 23.7 (CH3, C19), 24.8 (CH2,
C15), 27.4 (CH2, C7), 28.4 (CH2, C6), 28.7 (CH2, C16), 29.9 (CH2, C11), 31.1 (CH2, C2), 33.0 (CH2,
C23), 33.8 (CH2, C22), 34.8 (CH, C9), 36.9 (CH), 37.2 (CH), 37.4 (CH), 43.6 (CH, C5), 47.5 (CH,
- 133 -
C17), 48.0 (CH, C14), 63.0 (CH2), 63.1 (CH2), 67.9 (CH2), 68.0 (CH2) 72.5 (CH, C3), 74.0 (CH2,
C12), 126.1 (=CH2), 129.8 (=CH-), 177.4 (CO, C24) ppm.
IR= 3415 (NH), 3245 (OH), 2925 (alkyl), 2852 (alkyl), 1646 (C=O), 1441, 1364, 1292 cm-1
.
MS (+ESI) m/z= Found 503.4202 (M-Br)+; calculated for C31H55N2O3 503.4207; 1.0 ppm.
Preparation of 2-[[(4R)-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-
2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-
yl]pentanoyl]amino]ethyl-dimethyl-[(4-vinylphenyl)methyl]ammonium chloride (compound 109)
Compound 62 (0.2 g 0.0004 mol) was dissolved in chloroform (10 mL). Vinyl benzyl chloride (0.64
mL, 0.004 mol) was added and the solution was stirred overnight at ambient temperature. The
resulting precipitate was collected by vacuum filtration and was dried overnight under vacuum. The
product was an off-white solid. TLC 100% MeOH, Rf 0.1 (single spot).
Yield; 0.07 g, 0.0016 mol, 26.9 %.
Melting point: 143.8 – 148.9 oC.
1H NMR (MeOD) (250 MHz) δ= 0.66 (s, 3H, 18-CH3), 0.94 (s, 3H, 19-CH3), 1.10-2.38 (m, 33H,
steroidal backbone CH/CH2), 3.09 (s, 6H, 2x CH3), 3.41 (t, 2H, CH2, J= 7.5), 3.73 (t, 2H, CH2, J=
- 134 -
5.0), 4.56 (s, 2H, CH2), 5.37 (d, 1H, =CH-, J = 12.5), 5.91 (d, 1H, =CH, J= 20.0), 6.80 (dd, 1H, =CH,
J= 10.0 and 17.5 ), 7.57 (dd, 4H, Ar-CH, J= 5.0) ppm.
13C NMR (MeOD) (62.9 MHz) δ= 11.7 (CH3, C18), 18.0 (CH3, C21), 21.1 (CH2, C11), 23.1 (CH3,
C19), 24.4 (CH2, C15), 26.8 (CH2, C7), 27.5 (CH2, C6), 28.4 (CH2, C16), 30.3 (CH2, C2), 33.0 (CH2),
33.9 (CH2), 35.7 (CH2), 36.8 (CH), 37.2 (CH), 41.9 (CH), 43.5 (CH), 57.4 (CH, C17), 57.9 (CH, C14),
63.5 (CH2), 71.6 (CH, C3), 115.8 (=CH2), 126.9 (Ar-CH), 127.2 (Ar-CH), 133.6 (Ar-CH), 136.2 (Ar-
C), 140.8 (=CH-), 176.5 (CO, C24) ppm.
IR= 3339 (NH), 3215 (OH), 2921 (alkyl), 2857 (alkyl), 1667 (C=O), 1646, 1445, 1356, 1070 (R2CH-
OH) cm-1
.
MS (+ESI) m/z= Found 563.4558 (M-Cl)+; calculated for C37H59N2O2 563.4571; 2.3 ppm.
Preparation 3-[[(4R)-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-
2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-
yl]pentanoyl]amino]propyl-trimethyl-ammonium iodide (compound 110).
Compound 64 (0.5 g 0.002 mol) was dissolved in solution of chloroform (15 mL) and methanol (4
mL). Iodomethane (3.63 mL, 0.02 mol) was added and the solution was stirred for four days at
ambient temperature. To induce precipitation the solution was placed in an acetone/dry ice mixture (-
78 oC). The resulting precipitate was collected by vacuum filtration and was dried overnight under
vacuum. The product was an off-white solid. TLC 100% MeOH Rf 0.1 (single spot).
- 135 -
Yield; 0.42 g, 0.06 mol, 64.6 %.
Melting point: 253.8-255.4 oC.
1H NMR (CDCl3) (250 MHz) δ= 0.69 (s, 3H, 18-CH3), 0.94 (s, 3H, 19-CH3), 1.00-2.38 (m, 33H,
steroidal backbone CH/CH2), 3.13 (s, 9H, 3 x CH3), 3.54 (m, 1H, 3-CH) ppm.
13C NMR (CDCl3) (62.9 MHz) δ= 10.9 (CH3, C18), 17.3 (CH3, C21), 20.4 (CH2, C11), 22.4 (CH2),
23.0 (CH3, C19), 23.7 (CH2, C15), 26.1 (CH2, C7), 26.8 (CH2, C6), 28.0 (CH2, C16), 29.6 (CH2, C2),
31.6 (CH2, C22), 32.5 (CH2, C23), 34.1 (C, C10), 34.9 (CH, C20), 35.4 (CH2, C1), 35.6 (CH, C8),
35.7 (CH2, C4), 40.0 (CH2, C12), 40.4 (CH, C9), 42.0 (CH, C5), 42.4 (C, C13), 52.2 (CH, C17), 55.8
(CH, C14), 56.8 (CH), 64.2 (CH2), 70.8 (CH, C3), 175.7 (CO, C24) ppm.
IR= 3386 (OH), 2921 (alkyl), 2852 (alkyl), 1641 (C=O), 1552, 1441, 1368, 1253 (C=O ester stretch),
1031 (R2CH-OH) cm-1
.
MS (+ESI) m/z= Found 475.4256 (M-I)+; calculated for C30H55N2O2 475.4258; 0.4 ppm.
Preparation of 3-[[(4R)-4-[(3R,10S,12S,13R,17R)-3,12-dihydroxy-10,13-dimethyl-
2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-
yl]pentanoyl]amino]propyl-trimethyl-ammonium iodide (compound 111)
Compound 65 (0.1 g 0.0002 mol) was dissolved in chloroform (5 mL). Iodomethane (0.29 mL, 0.002
mol) was added and the solution was stirred overnight at ambient temperature. The resulting
- 136 -
precipitate was collected by vacuum filtration and was dried overnight under vacuum. The product
was an off-white solid. TLC 100% MeOH Rf 0.1 (single spot).
Yield; 0.09 g, 0.0014 mol, 75 %.
Melting point: 150.1 – 153.3 oC.
1H NMR (MeOD) (250 MHz) δ= 0.71 (s, 3H, 18-CH3), 0.93 (s, 3H, 19-CH3), 1.03 (d, 3H, 21- CH3, J=
7.5), 1.05-2.38 (m, 33H, steroidal backbone CH/CH2), 3.15 (s, 6H, 2 x CH3), 3.53 (m, 1H, 3-CH), 3.95
(s, 1H, 12-CH) ppm.
13C NMR (MeOD) (62.9 MHz) δ= 13.2 (CH3, C18), 17.7 (CH3, C21), 23.7 (CH3, C19), 27.4 (CH2,
C7), 28.4 (CH2, C6), 28.7 (CH2, C16), 29.9 (CH2, C11), 31.1 (CH2, C2), 33.1 (CH2, C23), 35.3 (C,
C10), 37.2 (CH2, C4), 43.6 (CH, C5), 72.5 (CH, C3), 74.0 (CH2, C12), 177.3 (CO, C24) ppm.
IR= 3364 (OH), 2933 (alkyl), 2852 (alkyl), 1654 (C=O), 1548, 1437 cm-1
.
MS (ES) m/z= Found 491.4195 (M-I)+; calculated for C30H55N2O3 491.4207; 2.5 ppm.
- 137 -
Preparation of allyl-[3-[[(4R)-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-
2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-
yl]pentanoyl]amino]propyl]-dimethyl-ammonium bromide (compound 112)
Compound 64 (0.5 g 0.001 mol) was dissolved in dichloromethane (20 mL). Allyl bromide (1.88 mL,
0.01 mol) was added and the solution was stirred for a further 5 days. No precipitate had been formed
so the vial was cooled to -78 oC and a precipitate formed and collected by vacuum filtration. TLC
100% MeOH Rf 0.1 (single spot).
Yield; 0.45 g, 0.0007 mol, 71 %.
Melting point: 191.1 – 209.9 oC.
1H NMR (CDCl3) (250 MHz) δ= 0.68 (s, 3H, 18-CH3), 0.94 (s, 3H, 19-CH3), 1.00-2.38 (m, 33H,
steroidal backbone CH/CH2), 3.10 (s, 6H, 2 x CH3), 3.27 (q, 2H, CH2), 3.54 (m, 1H, 3-CH), 4.01 (d,
2H, CH2, J= 7.5), 5.74 (t, 2H, CH2, J= 10.0), 6.08 (m, 1H, =CH-) ppm.
13C NMR (CDCl3) (62.9 MHz) δ= 11.1 (CH3, C18), 17.5 (CH3, C21), 20.5 (CH2, C11), 22.5 (CH3,
C19), 23.8 (CH2, C15), 26.2 (CH2, C7), 26.9 (CH2, C6), 27.0 (CH2, C16), 29.7 (CH2, C2), 31.7 (CH2,
C22), 32.5 (CH2, C23), 34.2 (C, C10), 35.0 (CH, C20), 35.4 (CH2, C1), 35.7 (CH, C8), 35.9 (CH2,
C4), 40.0 (CH2, C12), 40.4 (CH, C9), 42.0 (CH, C5), 42.4 (C, C13), 55.9 (CH, C17), 56.4 (CH, C14),
61.7 (CH2), 66.1 (CH2), 70.9 (CH, C3), 124.8 (=CH2), 128.0 (=CH-), 175.8 (CO, C24) ppm.
- 138 -
IR= 3411 (NH), 3270 (OH), 2925 (alkyl), 2857 (alkyl), 1646 (C=O), 1543, 1437, 1373, 1036 (R2CH-
OH) cm-1
.
MS (+ESI) m/z= Found 501.4402 (M-Br)+; calculated for C31H55N2O2 501.4415: 2.5 ppm.
Preparation of cyclopentylmethyl-[3-[[(4R)-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-
2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-
yl]pentanoyl]amino]propyl]-dimethyl-ammonium bromide (compound 113)
Compound 64 (0.5 g 0.001 mol) was dissolved in chloroform (20 mL) with benzyl bromide (1.37 mL,
0.008 mol) and the solution was stirred for 48 hours. The solvent was evaporated under reduced
pressure, then re-dissolved in methanol and washed with petroleum ether 60/80 (3 x 20 mL). The
solvent was removed under reduced pressure to produce a white powder. TLC 100% MeOH Rf 0.1
(single spot).
Yield; 0.24 g, 0.0003 mol, 35 %.
Melting point: 145-147.9 oC.
1H NMR (MeOD) (250 MHz) δ= 0.68 (s, 3H, 18-CH3), 0.94 (s, 3H, 19-CH3), 1.00-2.38 (m, 33H,
steroidal backbone CH/CH2), 3.05 (s, 6H, 2 x CH3), 3.54 (m, 1H, 3-CH), 4.56 (s, 2H, CH2), 7.55
(broad s, 5H, Ar-CH) ppm.
- 139 -
13C NMR (MeOD) (62.9 MHz) δ= 10.9 (CH3, C18), 17.3 (CH3, C21), 20.4 (CH2, C11), 22.4 (CH3,
C19), 22.6 (CH2, C15), 23.7 (CH), 26.1 (CH2, C7), 26.8 (CH2, C6), 27.7 (CH2, C16), 29.6 (CH2, C2),
31.6 (CH2, C22), 32.5 (CH2, C23), 34.1 (C, C10), 34.9 (CH, C20), 35.3 (CH2, C1), 35.6 (CH, C8),
35.6 (CH2, C4), 39.9 (CH2, C12), 40.3 (CH, C9), 41.9 (CH, C5), 42.3 (C, C13), 55.8 (CH, C17), 56.3
(CH, C14), 61.7 (CH), 67.4 (CH), 70.8 (CH, C3), 127.3 (Ar-CH), 128.8 (Ar-CH), 130.3 (Ar-CH),
132.6 (Ar-C), 175.6 (CO, C24) ppm.
IR= 3253 (OH), 2929 (alkyl), 2865 (alkyl), 1637 (C=O), 1565, 1441 cm-1
.
MS (+ESI) m/z= Found 551.4570 (M-Br)+; calculated for C36H59N2O2 551.4571; 0.2 ppm.
Preparation of 3-[[(4R)-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-
2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-
yl]pentanoyl]amino]propyl-dimethyl-[(4-vinylphenyl)methyl]ammonium chloride (compound
114)
Compound 64 (1.0 g 0.001 mol) was dissolved in dichloromethane (20 mL).Vinyl benzyl chloride
(0.89 mL, 0.005) was added and the solution was stirred for 48 hours at ambient temperature. The
resulting precipitate was collected by vacuum filtration and was washed with petroleum ether 60/80 (3
- 140 -
x 20 mL). The resultant crude product was dissolved in methanol and again washed with petroleum
ether 60/80 (3 x 20 mL). Solvent removed under reduced pressure to produce off-white solid. TLC
100% MeOH Rf 0.1 (single spot).
Yield; 1 g, 0.001 mol, 75 %.
Melting point: > 350 oC.
1H NMR (MeOD) (250 MHz) δ= 0.67 (s, 3H, 18-CH3), 0.94 (s, 3H, 19-CH3), 1.00-2.38 (m, 33H,
steroidal backbone CH/CH2), 3.05 (s, 6H, 2 x CH3), 4.54 (s, 2H, CH2), 5.37 (d, 1H, =CH-, J= 12.5),
5.91 (d, 1H, =CH, J=15.0), 6.80 (dd, 1H, =CH, J= 12.5 and 17.5), 7.56 (dd, 5H, Ar-CH, J= 7.5) ppm.
13C NMR (MeOD) (62.9 MHz) δ= 11.9 (CH3, C18), 18.2 (CH3, C21), 20.6 (CH2, C11), 23.3 (CH3,
C19), 24.0 (CH2, C15), 26.3 (CH2, C7), 27.1 (CH2, C6), 28.0 (CH2, C16), 30.3 (CH2, C2), 30.9 (CH2,
C22), 30.9 (CH2, C23), 34.2 (C, C10), 35.1 (CH, C20), 35.3 (CH2, C1), 35.6 (CH, C8), 36.3 (CH2,
C4), 40.0 (CH2, C12), 40.2 (CH, C9), 41.9 (CH, C5), 42.4 (C, C13), 55.8 (CH, C17), 56.3 (CH, C14),
70.5 (CH, C3), 178.1 (CO, C24) ppm.
IR= 3356 (OH), 2929 (alkyl), 2852 (alkyl), 1633 (C=O), 1548, 1441, 1031 (R2CH-OH) cm-1
.
MS (+ESI) m/z= Found 577.4723 (M-Cl)+; calculated for C38H61N2O2 577.4728; 0.8 ppm.
- 141 -
Preparation of (4R)-4-[(3R,10S,12S,13R,17R)-3,12-dihydroxy-10,13-dimethyl-
2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]-N-[2-(1-
methylpyrrolidin-1-ium-1-yl)ethyl]pentanamide iodide (compound 115)
Compound 76 (0.1 g 0.0002 mol) was dissolved in chloroform (10 mL). Methyl iodide (0.09 mL
0.0006 mol) was added and the solution was stirred for 1 week. No precipitate formed so solvent was
evaporated under reduced pressure. Material was re-dissolved in methanol and washed with petroleum
ether 60/80 (3 x 20 mL). Green/brown oil produced. TLC 100% MeOH Rf 0.1 (single spot).
Yield; 0.08 g, 0.0001mol, 66 %.
Melting point: Oil.
1H NMR (CDCl3) (250 MHz) δ= 0.70 (s, 3H, 18-CH3), 0.93 (s, 3H, 19-CH3), 1.00 (d, 3H, 21-CH3 J=
10.0), 1.20-2.38 (m, 33H, steroidal backbone CH/CH2), 3.15 (s, 3H, CH3), 3.50-3.70 (multiple
overlapping multiplets, 7H, CH/CH2), 3.95 (s, 1H, 12-CH) ppm.
13C NMR (CDCl3) (250 MHz) δ= 13.2 (CH3, C18), 17.7 (CH3, C21), 22.56 (CH2), 23.7 (CH3, C19),
24.9 (CH2, C15), 27.5 (CH2, C7), 28.44 (CH2, C6), 28.7 (CH2, C16), 29.9 (CH2, C11), 31.1 (CH2, C2),
33.0 (CH2, C23), 33.9 (CH2, C22), 34.8 (CH, C9), 35.3 (C, C10), 36.4 (CH2, C1), 36.9 (CH, C20),
37.2 (CH2, C4), 37.4 (CH, C8), 43.6 (CH, C5), 63.5 (CH2), 66.1 (CH2), 72.5 (CH, C3), 74.0 (CH2,
C12), 176.5 (CO, C24) ppm.
- 142 -
IR= 3394 (OH), 2916 (alkyl), 2857 (alkyl), 1646 (C=O), 1530, 1445, 1368, 1253, 1036 (RCH-OH)
cm-1
.
MS (+ESI) m/z= Found 503.4201 (M-I)+; calculated for C31H55N2O3 503.4207; 1.2 ppm.
Preparation of (4R)-N-[2-(1-allylpyrrolidin-1-ium-1-yl)ethyl]-4-[(3R,10S,13R,17R)-3-hydroxy-
10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-
17-yl]pentanamide bromide (compound 116)
Compound 75 (0.5 g 0.001 mol) was dissolved in dichloromethane (15 mL). Ally bromide (2.79 mL,
0.02) was added and the solution was stirred for 48 hours at 50 oC. The resulting precipitate was
collected by vacuum filtration and was triturated chloroform (3 x 20 mL). TLC 100% MeOH Rf 0.1
(single spot).
Yield; 0.168 g, 0.0002 mol, 27 %.
Melting point: 211.6- 214.8 oC.
1H NMR (MeOD) (250 MHz) δ= 0.68 (s, 3H, 18-CH3), 0.94 (s, 3H, 19-CH3), 1.00-2.38 (m, 33H,
steroidal backbone CH/CH2), 3.62 (overlapping mutliplets, 4H, CH2), 4.02 (d, 2H, CH2, J= 7.5), 5.70-
5.79 (multiple overlapping multiplets, 2H, =CH2), 6.11 (m, 1H, =CH-) ppm.
- 143 -
13C NMR (MeOD) (62.9 MHz) δ= 11.0 (CH3, C18), 17.3 (CH3, C21), 20.4 (CH2, C11), 22.3 (CH3,
C19), 22.4 (CH), 23.7 (CH2, C15), 26.1 (CH2, C7), 26.8 (CH2, C6), 27.7 (CH2, C16), 29.6 (CH2, C2),
31.4 (CH2, C22), 32.3 (CH2, C23), 33.2 (C, C10), 34.1 (CH, C20), 34.9 (CH2, C1), 35.3 (CH, C8),
35.6 (CH2, C4), 40.0 (CH2, C12), 40.3 (CH, C9), 42.0 (CH, C5), 42.4 (C, C13), 55.6 (CH, C17), 56.4
(CH, C14), 57.8 (CH2), 61.3 (CH2), 62.1 (CH2), 70.8 (CH, C3), 125.2 (=CH2), 127.3 (=CH-), 175.8
(CO, C24) ppm.
IR= 3411 (NH), 3198 (OH), 2925 (alkyl), 2852 (alkyl), 1637 (C=O), 1560, 1441, 1368, 1066 (R2CH-
OH) cm-1
.
MS (+ESI) m/z= Found 513.4405 (M-Br)+; calculated for C33H57N2O2 513.4415; 1.9 ppm.
Preparation of (4R)-N-[2-(1-allylpyrrolidin-1-ium-1-yl)ethyl]-4-[(3R,10S,12S,13R,17R)-3,12-
dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-
cyclopenta[a]phenanthren-17-yl]pentanamide bromide (compound 117)
Compound 76 (0.1 g 0.0002 mol) was dissolved in chloroform (10 mL) with allyl bromide (0.08 mL,
0.0006 mol). The solution was stirred for 1 week. The solvent was evaporated under reduced pressure
and the product was re-dissolved in methanol and washed with petroleum ether (3 x 10 mL). The
solvent was then removed under reduced pressure to produce a white solid. TLC 100% MeOH, Rf 0.1
(single spot).
Yield; 0.1 g, 0.0001 mol, 83 %.
- 144 -
Melting point:176.2 – 179.8 oC.
1H NMR (MeOD) (250 MHz) δ= 0.70 (s, 3H, 18-CH3), 0.93 (s, 3H, 19-CH3), 1.02 (d, 3H, 21-CH3, J=
5.0), 1.04-2.38 (m, 33H, steroidal backbone CH/CH2), 2.22 (broad s, 4H, CH2), 3.515 (q, 2H, CH2),
3.65 (m, 3H, CH2/3-CH), 4.01 (broad s, 1H, 12-CH), 4.025 (d, 2H, CH2 J= 7.5), 5.74 (m, 2H, =CH2),
6.11(m, 1H, =CH-) ppm.
13C NMR (MeOD) (62.9 MHz) δ= 12.4 (CH3, C18), 16.8 (CH3, C21), 21.8 (CH2), 22.9 (CH3, C19),
24.0 (CH2, C15), 26.6 (CH2), 27.6 (CH2, C7), 27.9 (CH2, C6), 28.1 (CH2, C16), 30.9 (CH2, C2), 32.2
(CH2, C23), 33.0 (CH2, C22), 34.0 (CH, C9), 36.0 (CH2), 36.6 (CH, C20), 42.7 (CH, C5), 44.8 (C,
C13), 47.1 (CH, C17), 58.4 (CH2), 61.9 (CH2), 62.8 (CH2), 71.7 (CH, C3), 73.1 (CH2, C12), 126.7
(CH2), 128.9 (CH2), 177.4 (CO, C24) ppm.
IR= 3305 (OH), 2921 (alkyl), 2857 (alkyl), 1641 (C=O), 1539, 1449, 1040 (R2CH-OH) cm-1
.
MS (+ESI) m/z= Found 529.4349 (M-Br)+; calculated for C33H57N2O3, 529.4364; 2.8 ppm.
Preparation of (4R)-N-[2-(1-benzylpyrrolidin-1-ium-1-yl)ethyl]-4-[(3R,10S,13R,17R)-3-hydroxy-
10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-
17-yl]pentanamide bromide (compound 118)
Compound 76 (0.5 g 0.001 mol) was dissolved in dichloromethane (20 mL) with benzyl bromide (1.42
mL 0.008 mol) was added and the solution was stirred for 24 hours. The resultant precipitate was
collected by vacuum filtration to produce a white powder. TLC 100% MeOH, Rf 0.1 (single spot).
- 145 -
Yield; 0.01 g, 0.0001 mol, 1.5 %.
Melting point: 216.2-220.8 oC.
1H NMR (CDCl3) (250 MHz) δ= 0.66 (s, 3H, 18-CH3), 0.94 (s, 3H, 19-CH3), 1.00-2.38 (m, 33H,
steroidal backbone CH/CH2), 3.54-3.76 (multiple overlapping multiplets, 7H, 3-CH/CH2), 4.57 (s, 2H,
CH2), 7.56 (m, 5H, Ar-CH) ppm.
13C NMR (CDCl3) (62.9 MHz) δ= 10.9 (CH3, C18), 17.3 (CH3, C21), 20.4 (CH2), 20.7 (CH2, C11),
22.4 (CH3, C19), 23.7 (CH2, C15), 27.7 (CH2, C16), 29.6 (CH2, C2), 31.5 (CH2, C22), 32.3 (CH2,
C23), 34.1 (C, C10), 34.9 (CH, C20), 35.3 (CH2, C1), 35.6 (CH, C8), 35.7 (CH2, C4), 40.0 (CH2,
C12), 40.3 (CH, C9), 41.9 (CH, C5), 42.4 (C, C13), 55.8 (CH, C17), 56.4 (CH, C14), 57.0 (CH), 61.2
(CH), 70.8 (CH, C3), 127.7 (Ar-CH), 129.0 (Ar-CH), 130.4 (Ar-CH), 132.2 (Ar-C), 175.8 (CO, C24)
ppm.
IR= 3343 (NH), 3241 (OH), 2929 (alkyl), 2844 (alkyl), 1667 (C=O), 1535, 1445, 1360, 1070 (R2CH-
OH) cm-1
.
MS (+ESI) m/z= Found 563.4563 (M-Br)+; calculated for C37H59N2O2 563.4571; 1.4 ppm.
- 146 -
Preparation of (4R)-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-
2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]-N-[2-[1-
[(4-vinylphenyl)methyl]pyrrolidin-1-ium-1-yl]ethyl]pentanamide chloride (compound 119)
Compound 75 (0.5 g 0.001 mol) was dissolved in dichloromethane (20 mL) with vinyl benzyl chloride
(1.74 mL 0.01 mol) was added and the solution was stirred for 48 hours. No precipitate formed so
solution was heated at 50 oC
overnight.
No precipitate formed so solvent was evaporated under
reduced pressure. Material re-dissolved in methanol and washed with petroleum ether 60/80 (3 x 20
mL). White solid produced. TLC 100% MeOH Rf 0.1 (single spot).
Yield: 0.08 g, 0.0001 mol, 12 %.
Melting point: >350 oC.
1H NMR (CDCl3) (250 MHz) δ= 0.65 (s, 3H, 18-CH3), 0.94 (s, 3H, 19-CH3), 1.00-2.24 (m, 33H,
steroidal backbone CH/CH2), 3.56 (multiple overlapping multiplets, 4H, CH2), 4.57 (s, 2H, CH2), 5.37
(d, 1H, =CH-, J= 12.5), 5.90 (d, 1H, =CH, J= 17.5), 6.80 (dd, 1H, =CH, J= 12.5 and 17.5), 7.60 (s, 4H,
Ar-CH) ppm.
13C NMR (CDCl3) (62.9 MHz) δ= 10.9 (CH3, C18), 17.3 (CH3, C21), 20.4 (CH2, C11), 20.7 (CH2),
22.4 (CH3, C19), 23.7 (CH2, C15), 26.1 (CH2, C7), 27.7 (CH2, C16), 29.6 (CH2, C2), 31.5 (CH2, C22),
32.3 (CH2, C23), 33.2 (C, C10), 34.1 (CH, C20), 34.9 (CH2, C1), 35.3 (CH, C8), 35.7 (CH2, C4), 40.0
(CH2, C12), 40.3 (CH, C9), 41.9 (CH, C5), 42.4 (C, C13), 55.8 (CH, C17), 56.4 (CH, C14), 57.0
- 147 -
(CH2), 61.2 (CH), 61.8 (CH2), 70.8 (CH, C3), 115.0 (Ar-C), 126.6 (=CH), 132.5 (=CH-), 135.4 (Ar-
CH), 139.9 (Ar-CH), 175.8 (CO, C24) ppm.
IR= 3360 (OH), 2925 (alkyl), 2852 (alkyl), 1641 (C=O), 1539, 1437, 1368, 1036 (R2CH-OH) cm-1
.
MS (+ESI) m/z= Found 589.4719 (M-Cl)+; calculated for C39H61N2O2 589.4728; 1.5 ppm.
Preparation of poly 3-[[(4R)-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-
2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-
yl]pentanoyl]amino]propyl-dimethyl-[(4-sec-butylphenyl)methyl]ammonium chloride
(compound 120)
Compound 64 (2.25 g 0.004 mol) was dissolved in chloroform (10 mL). Poly (vinyl benzyl chloride)
60/40 mixture of 3- and 4- isomers (0.25 g, 0.001 mol) was added and the solution was stirred
overnight at 50 oC. The resulting precipitate was collected by vacuum filtration. The product was a
yellow solid which would not dissolve in DMSO, TFA, acetone, 1, 4 dioxane, water or pyridine.
Yield; 0.63 g, 0.001 mol, 20 %.
No NMR achieved due to insolubility of material. Deuterated solvents tried; chloroform, methanol,
dimethyl sulfoxide, H2O, trifluoroacetic acid, acetic acid, acetone, 1, 4-dioxane, pyridine and
dimethoxyethane. Presumed crosslinking of material has occurred, resulting in insolubility.
IR= 3270, (OH/NH), 2925 (alkyl), 2865 (alkyl), 1641 (C=O), 1543, 1441 cm-1
.
- 148 -
Preparation of poly 3-[[(4R)-4-[(3R,10S,12S,13R,17R)-3,12-dihydroxy-10,13-dimethyl-
2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-
yl]pentanoyl]amino]propyl-dimethyl-[(4-sec-butylphenyl)methyl]ammonium chloride
(compound 121)
Product from 2/90 (0.75 g 0.001 mol) was dissolved in chloroform (10 mL). Poly (vinyl benzyl
chloride) 60/40 mixture of 3- and 4- isomers (0.83 g, 0.005 mol) was added and the solution was
stirred overnight at ambient temperature. The resulting precipitate was collected by vacuum filtration
and then triturated in hot chloroform (3 x 20 mL). The product was an off-white solid.
Yield; 0.906 g, 0.001 mol, 81 %.
Melting point: >350 oC.
1H NMR (MeOD) (250 MHz) δ= 0.71 (broad s, 3H, 18-CH3), 0.94 (broad s, 3H, 19-CH3), 3.56 (broad
s, 1H, 3-CH), 3.97 (broad s, 1H, 12-CH), 6.55-7.15 (broad s, 4H, Ar-CH) ppm.
IR= 3317 (OH), 2921 (alkyl), 2852 (alkyl), 1641 (C=O), 1445, 1258 (C=O ester stretch), 1040 (R2CH-
OH) cm-1
.
- 149 -
Preparation of (4R)-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-
2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]-1-[4-(2-
hydroxyethyl)-4-methyl-piperazin-4-ium-1-yl]pentan-1-one iodide (compound 122).
Compound 94 (0.2 g 0.0004 mol) was dissolved in chloroform (5 mL). Iodomethane (0.29 mL, 0.002
mol) was added and the solution was stirred overnight at ambient temperature. The resulting
precipitate was collected by vacuum filtration and was dried overnight under vacuum. The product
was an off-white solid. TLC 100% MeOH Rf 0.1 (single spot).
Yield; 0.01 g, 0.00001 mol, 4 %.
Melting point: 245.3 – 251.3 oC.
1H NMR (MeOD) (250 MHz) δ= 0.69 (s, 3H, 18-CH3), 0.94 (s, 3H, 19-CH3), 0.98 (d, 3H, 21-CH3, J=
5.0), 1.00-2.55 (m, 33H, steroidal backbone CH/CH2), 3.54-3.65 (multiple overlapping multiplets, 6H,
CH2), 3.97- 4.05 (multiple overlapping multiplets, 6H, CH2) ppm.
13C NMR (MeOD) (62.9 MHz) δ= 11.5 (CH3, C18), 16.4 (CH3, C21), 22.9 (CH2, C11), 25.9 (CH2),
27.8 (CH2), 32.2 (CH), 33.2 (CH), 34.6 (C, C10), 35.1 (CH, C20), 41.1 (CH, C5), 42.4 (C, C13), 44.5
(CH2), 45.8 (CH), 47.4 (CH), 60.4 (CH2), 61.3 (CH), 61.8 (CH), 67.1 (CH2), 70.1 (CH, C3), 71.2
(CH2), 75.7 (CH2), 76.3 (CH2), 80.6 (CH2), 174.8 (CO, C24) ppm.
IR= 3313 (OH), 2929 (alkyl), 2852 (alkyl), 1607 (C=O), 1466, 1249, 1044 (R2CH-OH) cm-1
.
- 150 -
MS (+ESI) m/z= Found 503.4195 (M-I)+; calculated for C31H55N2O3 503.4207; 2.4 ppm.
Preparation of (4R)-1-[4-allyl-4-(2-hydroxyethyl)piperazin-4-ium-1-yl]-4-[(3R,10S,13R,17R)-3-
hydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-
cyclopenta[a]phenanthren-17-yl]pentan-1-one bromide (compound 123)
Compound 94 (0.2 g 0.0004 mol) was dissolved in chloroform (5 mL). Allyl bromide (0.44 mL, 0.003
mol) was added and the solution was stirred overnight at ambient temperature. The resulting
precipitate was collected by vacuum filtration and was dried overnight under vacuum. The product
was an off-white solid. TLC 100% MeOH Rf 0.1 (single spot).
Yield; 0.02 g, 0.00003 mol, 8 %.
Melting point: 194.9 – 197.8 oC.
1H NMR (MeOD) (250 MHz) δ= 0.70 (s, 3H, 18-CH3), 0.94 (s, 3H, 19-CH3), 0.98 (d, 3H, 21-CH3, J=
7.5), 1.00-2.53 (m, 33H, steroidal backbone CH/CH2), 3.54-3.65 (multiple overlapping multiplets, 6H,
CH2), 4.01 (multiple overlapping multiplets, 6H, CH2), 4.27 (d, 2H, CH2, J= 7.5), 5.76 (m, 2H, =CH2),
6.10 (m, 1H, =CH-) ppm.
13C NMR (MeOD) (62.9 MHz) δ= 12.5 (CH3, C18), 18.9 (CH3, C21), 21.9 (CH2, C11), 23.9 (CH3,
C19), 25.3 (CH2, C15), 27.6 (CH2, C6), 28.3 (CH2, C16), 29.3 (CH2, C2), 30.6 (CH2, C22), 31.1 (CH2,
C23), 32.2 (C, C10), 35.6 (CH, C20), 36.4 (CH2, C1), 36.9 (CH, C8), 37.2 (CH2, C4), 41.5 (CH2,
- 151 -
C12), 41.9 (CH, C9), 56.4 (CH, C17), 57.4 (CH, C14), 57.9 (CH2), 59.4 (CH2), 61.6 (CH2), 63.7
(CH2), 72.4 (CH, C3), 125.6 (CH), 129.9 (CH), 174.8 (CO, C24) ppm.
IR= 3350 (NH), 3241 (OH), 2938 (alkyl), 2850 (alkyl), 1633 (C=O), 1439, 1244, 1189.78 (C=O ester
stretch), 1244 (R2CH-OH) cm-1
.
MS (ES) m/z= Found 529.4349 (M-Br)+; calculated for C33H57N2O3 529.4364; 2.8 ppm.
Preparation of (4R)-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-
2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]-1-[4-(2-
hydroxyethyl)-4-pentyl-piperazin-4-ium-1-yl]pentan-1-one bromide (compound 124)
Compound 64 (0.5 g 0.001 mol) was dissolved in dichloromethane (20 mL). Iodopentane (1.57 mL,
0.007 mol) was added and the solution was stirred overnight at 40 oC. The resulting precipitate was
collected by vacuum filtration and was dried overnight under vacuum. The product was an off-white
solid. TLC 100% MeOH Rf 0.1 (single spot).
Yield; 0.36 g, 0.0005 mol, 50.7 %.
Melting point: 104.8 – 108.6 oC.
- 152 -
1H NMR (MeOD) (250 MHz) δ= 0.66 (s, 3H, 18-CH3), 0.92 (s, 3H, 19-CH3), 0.94 (multiple
overlapping multiplets consisting of both CH3 and CH2), 1.00-2.38 (m, 33H, steroidal backbone
CH/CH2), 3.08 (s, 5H, CH3/CH2). 3.51 (m, 1H, 3-CH) ppm.
13C NMR (MeOD) (62.9 MHz) δ= 11.0 (CH3, C18), 12.7 (CH3), 17.3 (CH3, C21), 20.4 (CH2,
C11), 21.8 (CH3, C19), 26.1 (CH2, C7), 29.6 (CH2, C2), 34.1 (C, C10), 35.4 (CH, C20), 35.7
(CH2, C1), 40.0 (CH2, C12), 40.4 (CH, C9), 42.0 (CH, C5),42.4 (C, C13), 49.8 (CH), 55.9
(CH, C17), 56.4 (CH, C14), 70.8 (CH, C3), 175.7 (CO, C24) ppm.
IR= 3449 (OH), 2921 (alkyl), 2857 (alkyl), 1641 (C=O), 1548, 1445, 1364, 1036 (R2CH-OH) cm-1
.
MS (+ESI) m/z= Found 531.4880 M+; calculated for C34H63N2O2 531.4884; 0.8 ppm.
Preparation of (4R)-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-
2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]-1-[4-(2-
hydroxyethyl)-4-[(4-vinylphenyl)methyl]piperazin-4-ium-1-yl]pentan-1-one chloride (compound
125)
Compound 94 (0.2 g 0.0004 mol) was dissolved in chloroform (5 mL). Vinyl benzyl chloride (0.31
mL, 0.002 mol) was added and the solution was stirred overnight at ambient temperature. The
resulting precipitate was collected by vacuum filtration and was dried overnight under vacuum. The
product was an off-white solid. TLC 100% MeOH Rf 0.1 (single spot).
- 153 -
Yield; 0.1 g, 0.0001 mol, 38 %.
Melting point: 143.6- 146.0 oC.
1H NMR (MeOD) (250 MHz) δ= 0.680 (s, 3H, 18-CH3), 0.94 (s, 3H, 19-CH3), 1.00-2.53 (m, 33H,
steroidal backbone CH/CH2), 3.61 (multiple overlapping multiplets, 6H, CH2), 5.37 (d, 1H, =CH-, J=
12.5), 5.90 (d, 1H, =CH, J = 17.5), 6.80 (dd, 1H, =CH, J= 12.5 and 17.5), 7.59 (broad s, 4H, Ar-CH)
ppm.
13C NMR (MeOD) (62.9 MHz) δ= 12.5 (CH3, C18), 18.9 (CH3, C21), 21.9 (CH2, C11), 23.9 (CH3,
C19), 25.3 (CH2, C15), 27.6 (CH2, C7), 28.3 (CH2, C6), 29.3 (CH2, C16), 30.6 (CH2, C2), 31.2 (CH2,
C22), 32.2 (CH2, C23), 35.6 (C, C10), 36.4 (CH, C20), 36.8 (CH2, C1), 37.2 (CH, C8), 40.6 (CH2,
C12), 41.5 (CH, C9), 41.9 (CH, C5), 43.5 (C, C13), 56.5 (CH, C17), 57.4 (CH, C14), 57.9 (CH), 58.1
(CH2), 58.8 (CH2), 67.1 (CH), 72.4 (CH, C3), 116.7 (Ar-C), 127.2 (Ar-CH), 128.0 (Ar-CH), 134.9
(Ar-CH), 137.0 (=CH2), 141.6 (=CH-), 174.8 (CO, C24) ppm.
IR= 3283 (OH), 2925 (alkyl), 2857 (alkyl), 1616 (C=O), 1445, 1044 (R2CH-OH) cm-1
.
MS (ES) m/z= Found 605.4669 (M-Cl)+; calculated for C39H61N2O3 605.4677; 1.3 ppm.
- 154 -
2.3.4 Amino acid analogue synthesis
Preparation of methyl 2-(benzyloxycarbonylamino)-5-oxo-5-(4-vinylanilino)pentanoate
(compound 126)
From a modified experimental (Carlson et al., 2003) Z-Glutamic acid 1-methyl ester (1.0 g 0.003 mol)
was dissolved in dichloromethane (20 mL) with triethylamine (0.40 mL 0.003 mol), N-(3-
dimethylaminopropyl)-N’-ethyl carbodiimide hydrochloride (0.64 g, 0.003 mol), vinyl aniline (0.4
mL, 0.003 mol) and DMAP (0.001 g). The solution was left stirring overnight under argon conditions
at ambient temperature. The next day dichloromethane (30 mL) was added and the solution was
washed with sodium hydrogen carbonate (1 x 30 mL) and monobasic potassium phosphate (1 x 30
mL). Purified by column chromatography (100 % chloroform) for 200 mL then addition of 1 %
methanol. Solvent was evaporated under reduced pressure and precipitated by dissolving in methanol
and adding dropwise into stirring water. The resultant precipitate was collected by vacuum filtration,
washed with water (3 x 20 mL) and dried under vacuum.
Yield; ~20 mg (material dropped; only partially recovered).
Melting point: 145.3 – 147.7 oC.
- 155 -
1H NMR (CDCl3) (250 MHz) δ= 2.01-2.41 (overlapping multiplets, 4H, aliphatic CH2), 3.74 (s, 3H,
O-CH3), 4.44 (m, 1H, CH), 5.11 (s, 2H, CH2), 5.19 (d, 1H, CH, J= 10.0), 5.675 (d, 1H, CH, J= 17.5),
6.67 (dd, 1H, CH, J=12.5 and 17.5), 7.23-7.55 (overlapping multiplets, 9H, Ar-CH), 8.14 (broad
singlet, 1H, NH) ppm.
13C NMR (CDCl3) (62.9 MHz) δ= 29.6 (CH3) 33.8 (CH2), 52.6 (CH2), 53.3 (CH2), 67.3 (CH2), 112.9
(Ar-C), 119.7 (Ar-C), 126.8 (Ar-CH), 128.1 (Ar-CH), 128.3 (Ar-CH), 128.5 (Ar-CH), 133.6 (Ar-CH),
135.9 (Ar-CH), 136.1 (=CH2), 137.6 (=CH-), 156.7, 170.0, 172.2 (C=O) ppm.
IR= 3293 (NH), 1736 (C=O), 1684 (C=O), 1654 (C=O), 1269, 1241, 1211, 841 (aromatics), 689
(aromatics) cm-1
.
MS (ES) m/z= Found 397.1753 (M+H)+; calculated for C22H25N2O5 397.1758; 1.3 ppm.
Preparation of (2S)-2-(tert-butoxycarbonylamino)-6-(prop-2-enoylamino)hexanoic acid
(compound 127)
From literature (Li et al., 2013). Boc-Lys-OH (1 g, 0.004 mol) was dissolved in 10 mL (50/50 mixture
acetonitrile/2M NaOH) and was cooled to 10 oC. Acryloyl chloride (0.5 mL 0.005 mol) dissolved in 5
mL of acetonitrile was then added concurrently with NaOH (15 mL, 0.375 mol) to get the solution to
pH 10. Once the correct pH had been achieved, the solution was allowed to warm to ambient
temperature and left for a further 2 hours. After 2 hours the solution was acidified to just under pH 7
- 156 -
using 2 M HCl (15 mL, 0.41 mol) and extracted with ethyl acetate (3 x 20 mL). The organic layer was
dried over magnesium sulfate. Solvent was evaporated under reduced pressure and triturated with
diethyl ether to produce viscous oil.
Yield; 0.16 g, 0.0005 mol, 13 %.
Melting point: Oil.
1H NMR (MeOD) (250 MHz) δ= 1.44 (s, 9H, BOC), 3.22 (m, 2H, CH2), 4.05 (m, 1H, CH), 5.63 (dd,
1H, CH, J= 2.5 and 7.5), 6.22 (dd, 2H, CH2, J= 2.5 and 7.5) ppm.
13C NMR (MeOD) (62.9 MHz) δ= 28.2 (CH3 Boc), 32.6, 33.8, 36.4, 44.0 (CH2 lys-chain), 58.8 (CH),
84.4 (C, Boc), 101.4 (=CH2), 162.2 (C=O), 172.1 (C=O), 180.3 (C=O) ppm.
MS (ES) m/z= Found 323.1573 (M+H)+; calculated for C14H24N2O5Na 323.1577; 1.4 ppm.
2.3.5 Benzophenone derivatives
Preparation of N-(4-benzoylphenyl)acetamide (compound 128)
From a modification of (Bitonti, 1997) 4-aminobenzophenone (1.0 g 0.005 mol) was dissolved in
toluene (20 mL) with triethylamine (1.40 mL, 0.01 mol). Acetic anhydride (0.79 mL, 0.007 mol) was
then added. Once added, the solution was heated at reflux for 2 hours then added to water (50 mL)
wherein a precipitate was formed. The precipitate was collected by vacuum filtration, washed with
- 157 -
water (3 x 20 mL) and dried under vacuum. The crude product was then recrystallized from
acetonitrile to produce a white powder.
Yield; 0.41g, 0.0017 mol, 33.8 %.
Melting point: 153.7-155.2 oC. Lit 151-152
oC (Peet et al., 1989).
1H NMR (MeOD) (250 MHz) δ= 2.16 (s, 3H, CO-CH3), 7.52-7.76 (m, 9H, Ar-CH) ppm.
13C NMR (MeOD) (62.9 MHz) δ= 22.4 (CH3, COCH3), 118.5 (Ar-CH), 127.9 (Ar-CH), 129.2 (Ar-
CH), 130.9 (Ar-CH), 131.9 (Ar-CH), 142.9 (Ar-CH), 170.5 (CONH), 196.0 (Ar-CO) ppm.
IR= 3330 (NH), 1701 (C=O), 1513 (aromatic bending), 1407, 1253, 754 (aromatic) cm-1
.
MS (+ESI) m/z= Found 240.1022 (M+H)+; calculated for C15H14NO2 240.1024, 0.9 ppm.
Preparation of N, N’-bis(4-benzoylphenyl)decanediamide (compound 129)
Sebacoyl chloride (.34 mL g 0.001 mol) was dissolved in tetrahydrofuran (20 mL) with triethylamine
(0.40 mL 0.003 mol). 4-Aminobenzophenone (0.75 g 0.003 mol) and DMAP (~10 mg) was added and
the solution was stirred overnight at ambient temperature. Saturated sodium hydrogen carbonate
solution (50 mL) was then added and the solution extracted with ethyl acetate (3 x 20 mL). The
organic layers were combined and washed with 2 M hydrochloric acid (3 x 20 mL). The organic layer
was dried over magnesium sulfate. The solvent was evaporated under reduced pressure and the
product was recrystallized from ethanol to produce a white powder.
- 158 -
Yield: Not recorded.
Melting point: 192.8 – 201.6 oC.
1H NMR (250 MHz) (CDCl3) δ= 1.31 (s, 8H, aliphatic CH2), 1.61 (broad singlet, 4H, CH2), 2.35 (t,
4H, CH2, J= 7.5), 7.55-7.79 (overlapping multiplets, 18H, aromatics), 10.26 (s, 2H, NH) ppm.
13C NMR (CDCl3) (62.9 MHz) δ= 24.8, 28.5, 36.4, 38.4 (CH2, aliphatic chain), 118.1, 128.4, 129.3,
131.1, 137.5, 143.3 (CH, aromatic), 171.8 (C=O, amide), 194.4 (C=O, ketone) ppm.
IR= 3287 (NH), 2921 (alkyl), 1646 (C=O), 1586 (Aromatic C-H bending), 1398, 1309, 925
(aromatics) cm-1
.
MS (+ESI) m/z= Found 561.2740 (M+H)+; calculated for C36H37N2O4 561.2748; 1.2 ppm.
Preparation of N1, N3, N5-tris(4-benzoylphenyl)benzene-1,3,5-tricarboxamide (compound 130)
From a modification of (Jorgensen and Krebs, 2001) 1, 3, 5-benzenetricarbonyl trichloride (1.0 g
0.005 mol) was dissolved in tetrahydrofuran (20 mL). 4-aminobenzophenone (2.5 g 0.01 mol) was
added and the solution was stirred overnight. Solvent was evaporated under reduced pressure and
recrystallized from ethanol to produce a white powder.
- 159 -
Yield; 0.65 g, 0.0008 mol, 23 %.
Melting point: 281.4-283.4 oC.
1H NMR (d6-DMSO) (250 MHz) δ= 7.56-8.08 (overlapping multiplets, 27H, Ar-CH), 8.82 (s, 3H, Ar-
CH), 11.00 (s, 3H, NH) ppm.
13C NMR (d6-DMSO) (62.9 MHz) δ= 119.5, 128.5, 129.4, 130.3, 131.0, 132.0, 132.3, 135.1, 137.4,
143.0 (Ar-CH), 164.8, 194.63 (C=O) ppm.
IR= 3228, 3049 (NH), 1586, 1518, 1398, 1313, 848 (aromatic) cm-1
.
MS (+ESI) m/z= Found 748.2435 (M+H)+; calculated for C48H34N3O6 748.2442; 1.0 ppm.
2.4.1 Germination efficacy of sodium taurocholate solution/alternative.
The following is the protocol used by Christian Lowden, Kristian Poole and Amber Lavender for the
microbiological testing of the compounds synthesised;
A 2% (w/v) sodium taurocholate comprising double strength thioglycolate germination solution was
prepared as Wheeldon et al (2008). 200µL was added immediately, in triplicate, to 200µL C. difficile
NCTC 11204 spores from an original stock suspension of ~106cfu/mL in microfuge tubes and
vortexed. After 1 hour exposure, 600µL sterile distilled water was added to the test samples and
vortexed, and then subsequently heated in a water bath to 75°C for 20 minutes. Control samples
contained 200µL spore suspensions and 200µL sodium taurocholate germination solution, after 1 hour
exposure 600µL sterile distilled water was added to samples, vortexed and were subsequently stored
over ice for 20 minutes.
Following heat-shock, serial dilutions were performed and samples were inoculated onto Fastidious
Anaerobic Agar comprising 5% (v/v) defibrinated horse blood and 0.1% (w/v) sodium taurocholate.
Serial dilutions and inoculations were repeated for control samples.
- 160 -
Method adapted for alternative germination solution consisting 5mL DMSO and 5mL ‘Tween 20’ in
germination solution comprising 2% (w/v) sodium taurocholate and double strength thioglycolate
medium.
2.5.1 Unsuccessful Benzophenone reactions
The benzophenone derivatives were measured on a spectrofluorimeter and were found to have an
optimum excitation of 310 nm and a fluorescence emission of ~ 360 nm. The benzophenone bile acid
analogues (100 mg) were dissolved in chloroform (2 mL). From this solution, 20 μL was removed and
put in one well of a 96 well plate, which had previously been filled with a circular disc of PEEK
(Polyether ether ketone, fig 25) polymer of a radius to fit neatly into the well plate. The plate was then
exposed to 2.02 W/cm2 for 15 minutes, and then washed by sonication in chloroform 3 times. The
fluorescence of the material was then measured to see if any of the material had attached to the
polymer surface.
Fig 25: PEEK polymer (compound 131).
As no fluorescence of an appropriate wavelength was observed after trying to attach the bile amide
material to the polymer, this experiment was stopped. A similar set up was tried, with discs, but this
time different amounts of material was added (same concentration, 5-40 μL) and the polymer/bile
amide solutions were irradiated for differing periods of time, 15, 30 and 45 minutes. To ensure
solvents weren’t causing an effect, this method was repeated with the following solvents, ethyl acetate,
acetonitrile, tetrahydrofuran, ethanol and chloroform. None of these protocols produced any
131
- 161 -
measurable amount of attachment to the polymeric material, checked by using the spectrofluorimeter
to observe any emission.
- 162 -
3.0 Results and discussion:
3.1.1 Overview of project.
Scheme 10: Overall synthesis plan for project
- 163 -
In scheme 10, the overall outline for the project can be seen, broken down into distinct reaction
category steps. 1-Mixed anhydride amide bond formation; 2-esterification reaction; 3-aminolysis
amide bond formation; 4-Attachment of polymerizable groups to 3-OH position on bile ester; 5-
polymerization of attached polymerizable groups; 6-attachment of polymerizable groups to 3-OH on
bile amide; 7-polymerisation of attached polymerizable groups; 8-quaternization of tertiary amines; 9-
quaternization of tertiary amines using preformed polymeric alkylating materials.
Steroid based materials have been shown to have germinatory ability with regards to the hospital
acquired infection C. difficile. A set of structural analogues were produced to find an analogue of
taurocholate (a known germinant) which was also able to germinate C. difficile. That material was
then quaternized to produce a material which could possibly germinate the C. difficile spore and then
destroy the vegetative cell, as the vegetative cell is easier to kill.
As can be seen from scheme 10, there are many different synthetic routes which can be taken to
achieve the objective of producing a polymeric, potentially antimicrobial C. difficile germinating
material.
Some steps limited what could be achieved next, for example, once the material was quaternized, its
solubility changed considerably. Attaching an acryloyl group after quaternization would be virtually
impossible, due to the ionised material only being soluble in very polar solvents. There was also a
problem with the polymerisation of these ionised materials which had a potential polymer group in the
alkylating group. The way this was circumnavigated was to use preformed polymeric materials as the
alkylating agents.
There didn’t appear to be any difference in reactivity when attaching an acryloyl group to either the
bile amide or the methyl ester but it seemed easier to polymerise the ester version of the polymer
material than the amide version.
- 164 -
3.2.1Bile amide analysis
The most appropriate way to evaluate whether the synthesis of the steroidal material had been
successful was initially through TLC and then through use of proton NMR. The NMR had a tendency
to be very complicated due to the overlapping protons in the steroidal rings. However, key functional
groups were distinct and thus were able to be used as markers to indicate whether or not the reaction
had been successful or not.
It can be seen in fig 26 that there is considerable overlapping of signals in the 1-2 ppm range for bile
acids for a proton spectrum acquired at 250 MHz (Tamminen and Kolehmainen, 2001). What can be
done at the field strength available in this PhD project is to compare a known NMR spectrum of the
starting material (lithocholic and deoxycholic acid) with any potential product which may be formed,
or by comparison to known literature data relating to the three bile acids which were manipulated
(Waterhous et al., 1985).
There are key peaks in the proton NMR spectrum however, which are readily assignable and make it
easier to know whether the reaction has been successfully completed. When using deuterated
chloroform there are three methyl peaks in the parent steroid structure, two of which are singlets as
they are attached to the steroidal ring at quaternary carbons at positions 18/19, with an integration of
3H each. The final methyl peak is always a doublet as it is coming out from the ring at C21 and is
attached to a CH. Other key peaks include the multiplets around 3.6 ppm; this is from the hydrogen
that is attached at the 3 position on the steroidal ring, with the 7 position C-H (in deoxycholic) being a
much more compact singlet/triplet at 3.95 ppm (Hu et al., 2005). Depending on the type of bile amide
prepared, there will usually be a very “rounded” singlet, which is partially split into a poorly resolved
triplet at around 5.0-7.0 ppm. This is from the NH in the amide bond. The integration of the amide NH
can be compared to the methyl peaks and should be in a ratio of 1:3, suggesting formation of new
product. In the 13
C NMR spectrum there should only be one peak around the 170 ppm region, as this is
from the carbonyl group of the amide. If there is more than one peak present at 170ppm (with there
- 165 -
only being one in the expected structure) then it can be assumed that there is some starting material
left and that the material needs to be purified further.
An example of the complexity of the proton NMR is shown in fig 26. The top spectrum shows un-
modified lithocholic acid while the bottom spectrum is compound 102, which has an amide group and
an acryloylated 3-OH, thus showing some movements of the key peaks.
- 166 -
Fig 26: Proton NMR spectrum of lithocholic acid (top) and compound 102 (bottom). The horizontal
axis is in ppm.
There is a distinct shift in the 3-CH in the bottom NMR in fig 26. This peak, due to the attachment of
the electron-withdrawing acryloyl group has moved a significant distance from 3.5 ppm to 4.8 ppm.
Another signal which was an indicator of success was from the amide NH seen at 5.67 ppm on the
bottom NMR in fig 26. This peak was usually a broad singlet with shoulders, but wouldn’t always
- 167 -
appear in the same place. Both the proton NMR spectra show the complexity of the steroidal
hydrogens which is represented by overlapping peaks from ~1.06 to ~2.35. These peaks were
consistent and didn’t move.
Fig 27: Numbered carbons for compound 60.
Carbon number 13
C NMR CPD 62.9 MHz
(compound 60)
13C NMR 75.4 MHz Lithocholic
acid (Waterhous et al., 1985)
1 35.8 35.3
2 30.5 30.3
3 71.9 70.5
4 40.1 36.3
5 42.0 41.9
6 27.2 27.1
7 26.4 26.3
8 36.4 35.6
9 41.0 40.2
10 34.5 34.2
11 20.8 20.6
12 40.4 40.0
13 42.7 42.4
60
- 168 -
14 56.5 56.3
15 24.2 24.0
16 28.2 28.0
17 55.9 55.8
18 12.0 11.9
19 23.3 23.3
20 35.3 35.1
21 18.2 18.2
22 30.9 30.9
23 31.1 30.9
24 174.7 178.1
Table 3: Key peaks of 13
C NMR in both lithocholic acid and from compound 60 in CDCl3.
As can be seen from the table above, the interpretation of 13
C NMR can be very difficult, due to the
numerous peaks, which all have similar ppm values. The reference material found in (Waterhous et al,
1985) is particularly useful for assigning the new peaks on the compounds synthesised. Although
many of the peaks here are the same, when other, more complicated side chains are added, the
similarity drops, especially if the material becomes quaternized, thus making assignment of
quaternized materials very difficult. There can also be a significant variation depending on the
deuterated solvent used.
There have been many problems whilst trying to work in this particular area, particularly with initial
reactions focussed on the preparation of bile acid amides via aminolysis of the corresponding methyl
esters. As can be seen from the examples shown in the experimental, the aminolysis yields can be very
poor and the reaction times and conditions long and harsh. There does not seem at this point in time,
any particular rule as to why certain amides are able to be formed. As long as there is a primary amine,
the reaction should take place once a temperature of over 180 oC has been reached (to overcome
ammonium carboxylate salt formation). It was found however, with certain non-polar solvents
- 169 -
(toluene), that aminolysis could be achieved at lower temperature as non-polar solvents have a
tendency to disfavour salt formation (Allen et al., 2012). It was also noted, that aminolysis reactions
are more likely to work for lithocholic derivatives than for deoxycholic derivatives, and this is
presumably because lithocholic is less polar and therefore more soluble in non-polar solvents such as
toluene.
3.3.1 Solubility and reactivity of bile acids.
Due to the amphiphilic nature of the bile acids, solubility was often a key issue encountered when
trying to synthesise the bile amides for use in germination of C. difficile. The bile acids, particularly
deoxycholic acid were only soluble in a range of polar solvents. However, because of the coupling
methods used to produce the amides, alcohols were not an appropriate solvent system, so other
solvents had to be used, such as THF and 1,4-dioxane.
It had been reported that certain bile acids, under certain conditions could form gels in particular
solvents. Literature would suggest that lithocholic acid is more likely to form gels as opposed to
deoxycholic and cholic acid because the structure of lithocholic acid is relatively less polar than its
counterparts (Pal et al., 2009). Two bile acids can stay “attached” to each other through hydrogen
bonding on the carboxylic acid moiety on the bile acid. This was not clearly observed in any of the
reactions which were done in this project, but the same literature suggests that it is the ratio of bile
acid to amine which is very important.
When working with both lithocholic and deoxycholic derived bile amides, using the ethyl
chloroformate method to form the mixed anhydride, it was noted that upon addition of excess water to
the tetrahydrofuran or 1, 4-dioxane solution (dependent on bile acid used), that the majority of the
amides would precipitate from the aqueous THF/1, 4-dioxane solution and would require minimal
purification. It has been noted in the literature (Valkonen et al., 2008) however, that chenodeoxycholic
acid will not precipitate out in water. The only difference between this and deoxycholic acid is that the
hydroxyl is in the 7 position as opposed to the 12 position. This researcher suggests that it is the
- 170 -
positioning of the hydroxyl group in chenodeoxycholic (fig 28) acid which means it is more able to
form stable hydrogen bonds with the solution it is dissolved in than the deoxycholic acid, thus does not
precipitate out in polar solvents.
Fig 28: Chenodeoxycholic acid (compound 132).
One unusual observation was the differing solubility of the three bile acids worked on. It was found,
for the anhydride method, that lithocholic acid and cholic acid would dissolve readily in
tetrahydrofuran. Deoxycholic acid would not dissolve in tetrahydrofuran readily with the yields for the
products being significantly lower than if 1, 4-dioxane was used as the solvent. Even when dissolved
in the appropriate solvent, the inconsistencies between the yield of bile amide derivatives for both
lithocholic acid and deoxycholic acid were large. A specific example would be the formation of the
ester of lithocholic and deoxycholic acid with ethylene glycol (compounds 60 and 61). Even though
these two reactions were treated in exactly the same way the yields were significantly different.
Another situation where this difference is clearly visible is the conversion of the free acid to the
methyl ester. In the lithocholic acid reaction, the yield was 96.5 %, an excellent yield, yet, with the
deoxycholic version, the yield was 38 %, which is a relatively poor yield.
132
- 171 -
Fig 29: Two similar bile ester products, with very different yields, deoxycholic acid derivative
(compound 61) and lithocholic acid derivative (compound 60).
3.4.1 Preparation of bile acid esters
Scheme 11: Esterification of lithocholic acid
The starting material for the some of the reactions attempted was the preparation of the methyl ester of
both deoxycholic and lithocholic acid (scheme 11). This product was then used as a starting material
when proceeding with aminolysis reactions and also attachment of polymerizable groups at the 3-OH
position. This was achieved in a very straightforward, high yielding reaction (in lithocholic acid),
which took less than 1 hour to fully complete.
Attachment of ethylene glycol molecule to the carboxylic acid and therein producing an ester has been
successfully synthesised with both lithocholic acid and deoxycholic acid. It is a very easy reaction
with high yields and the product precipitates out of solution upon addition of water cleanly.
Unfortunately literature suggests that the incorporation of esters as opposed to amides into bile acids
nullifies any germinating ability the compounds may have (Howerton et al., 2010).
61 60
172
3.5.1 Aminolysis of bile acid esters, successes and failures.
Compound no.
Parent bile
acid
Attachment
position/
bond
formation
Amine Temperature
oC
Time Solvent Yield
62
Lithocholic
acid
Carboxylic
acid/amide
bond
100 24 hours toluene 69 %
63
Deoxycholic
acid
Carboxylic
acid/amide
bond
100 24 hours Toluene 24 %
64
Lithocholic
acid
Carboxylic
acid/amide
bond
140 24 hours 3-dimethylamino-
propylamine
69 %
65
Deoxycholic
acid
Carboxylic
acid/amide
bond
100 5 days 3-dimethylamino-
propylamine
73 %
173
Methyl
lithocholate
Methyl
ester/amide
bond
115 72 hrs. 2-(2-hydroxyethyl)
piperazine
N/A
Methyl
lithocholate
Methyl
ester/amide
bond
150 72 hrs. (2-aminoethyl)
piperidine
N/A
77
Methyl
lithocholate
Methyl
ester/amide
bond
150 48 hours (2-aminoethyl)
piperidine
1.6 %
78
Methyl
deoxycholate
Methyl
ester/amide
bond
70 5 days Methanol 45 %
Methyl
deoxycholate
Methyl
ester/amide
bond
150 72 hrs. Ethylene glycol N/A
174
Methyl
Lithocholate
Methyl
ester/amide
bond
150 72 hours Ethylene glycol N/A
Methyl
deoxycholate
Methyl
ester/amide
bond
150 48 hours 4-(aminoethyl)
piperadine
N/A
Methyl
lithocholate
Methyl
ester/amide
bond
150 24 hours 3-(dibutylamino)
propylamine
N/A
Methyl
deoxycholate
Methyl
ester/amide
bond
70 72 hours Methanol N/A
87
Methyl
deoxycholate
Methyl
ester/amide
bond
70 72 hours Methanol 25 %
88
Lithocholic
acid
Carboxylic
acid/methyl
ester
115 5 days Toluene 60 %
Methyl
deoxycholate
Methyl
ester/amide
bond
72 1 week Methanol N/A
175
Cholic acid Methyl
ester/amide
bond
115 96 hours Toluene N/A
Lithocholic
acid
Methyl
ester/amide
bond
115 96 hours Toluene N/A
Deoxycholic
acid
Methyl
ester/amide
bond
115 96 hours Toluene N/A
Cholic acid Methyl
ester/amide
bond
115 48 hours Toluene N/A
Cholic acid Methyl
ester/amide
bond
115 48 hours Toluene N/A
Deoxycholic
acid
Methyl
ester/amide
bond
115 72 hours Toluene N/A
Table 4: Showing all experiments tried using aminolysis methodology.
176
3.6.1 Aminolysis of bile acid esters.
Scheme 12: Aminolysis between methyl lithocholate and an amine.
Various methodologies were used to try and synthesise bile amides which would potentially be used as
compounds for the germination of C. difficile spores into vegetative cells, which are much easier to
kill. The first method of amid bond formation was through the use of aminolysis.
Aminolysis is the reaction of an ester with an amine to produce an amide and an alcohol as a side
product. With aminolysis, the prolonged reaction time that it takes to remove all of the starting
material usually meant that there were many side reactions going on. This made purification a problem
and indicates probable low yields.
With the reaction of methyl lithocholate and 4-(3-aminopropyl) morpholine, TLC indicated that all of
the starting material had been consumed. A proton NMR spectrum showed that there was an amide
bond present, yet there were so many other unexpected peaks, it was concluded that the reaction had
not worked.
When the reaction between methyl deoxycholate/lithocholate and 4-(aminoethyl) piperidine was
attempted, it was not successful. There were a large number of spots on the TLC plate after the
reaction had been worked up and thus was discontinued. Exactly the same thing happened for the
reaction between methyl lithocholate/deoxycholate and 3-(dibutylamino) propylamine. There does not
seem to be any clear reason as to why these specific examples did not work, especially as with some of
the reactions, the other analogue was successfully synthesised. Apart from the known problems with
177
deoxycholic acid, there could be problems with the nucleophilicity of the base or indeed the solubility
of the base being very different to that of the parent bile methyl ester.
The majority of the aminolysis reactions which used the reacting amine as the solvent did not work
(with the exception of compounds 64, 65 and 77). Aminolysis reactions were also carried out using
toluene as a solvent and there were more successes with this methodology relative to the number of
experiments tried using this methodology (compounds 62, 63, 88).
After the failures of these aminolysis reactions had occurred, it was suggested that if a water soluble
carbodiimide (N-(3-dimethylaminopropyl)-N’ethylcarbodiimide) was used, the reaction may work.
However, like the other carbonyldiimidazole reactions, this was unsuccessful. In this case there simply
was no reaction taking place at all, with no consumption of starting materials taking place. The reason
for this failure is not immediately clear but as there had been issues with solubility of the bile acids,
there may have been some solvent issues which affected the ability of the carbodiimide to form the
reactive intermediate.
178
3.7.1 Activation of bile acids and attempted amine coupling.
Table 5: Showing all reactions attempted using coupling reagent methodology.
Parent Bile
acid
Attachment
position/bond
formation
Amine Coupling agent Time Solvent Temperature oC
Yield Compound
number
Deoxycholic
acid
Carboxylic
acid/amide
bond
N,N’
carbonyldiimidazole
4 hours THF ambient N/A
Deoxycholic
acid
Carboxylic
acid/amide
bond
N,N’
carbonyldiimidazole
24 hours 50 N/A
179
Deoxycholic
acid
Carboxylic
acid/amide
bond
N,N’
carbonyldiimidazole
4 hours DMF Ambient N/A
Lithocholic
acid
Carboxylic
acid/amide
bond
Ethyl chloroformate 24 hours THF Ambient 33 %
96
Deoxycholic
acid
Carboxylic
acid/amide
bond
Ethyl chloroformate 48 hours THF Ambient 4 %
97
Lithocholic
acid
Carboxylic
acid/amide
bond
Ethyl chloroformate 24 hours THF Ambient 23.9 %
98
180
Deoxycholic
acid
Carboxylic
acid
Ethyl chloroformate 48 hours 1, 4
dioxane
Ambient 70 %
99
Lithocholic
acid
Carboxylic
acid/amide
bond
Ethyl chloroformate 24 hours THF Ambient N/A
Lithocholic
acid
Carboxylic
acid/amide
bond
Ethyl chloroformate 48 hours THF Ambient 43 %
94
Deoxycholic
acid
Carboxylic
acid/amide
bond
Ethyl chloroformate 48 hours 1, 4
dioxane
Ambient 46.8 %
95
Lithocholic
acid
Carboxylic
acid/amide
bond
Ethyl chloroformate 24 hours THF Ambient 23.5 %
66
181
Deoxycholic
acid
Carboxylic
acid/amide
bond
Ethyl chloroformate 24 hours 1,4
dioxane
Ambient 6 %
67
Lithocholic
acid
Carboxylic
acid/amide
bond
Boc-Lys-OH Ethyl chloroformate 24 hours THF Ambient 24 %
68
Deoxycholic
acid
Carboxylic
acid/amide
bond
Boc-Lys-OH Ethyl chloroformate 24 hours THF Ambient 11 %
69
Lithocholic
acid
Carboxylic
acid/amide
bond
Ethyl chloroformate 24 hours THF Ambient N/A
Compound
88
Amine/amide
bond
Methacrylic anhydride 72 hours Anhydr
ous
DCM
Ambient N/A
Lithocholic
acid
Carboxylic
acid/amide
bond
Ethyl chloroformate 24 hours THF Ambient 51 %
70
182
Deoxycholic
acid
Carboxylic
acid/amide
bond
Ethyl chloroformate 24 hours THF Ambient 42.8 %
71
Lithocholic
acid
Carboxylic
acid/amide
bond
Ethyl chloroformate 24 hours THF Ambient 51 %
93
Lithocholic
acid
Carboxylic
acid/amide
Ethyl chloroformate 24 hours THF Ambient 4 %
83
Lithocholic
acid
Carboxylic
acid/amide
bond
Ethyl chloroformate 24 hours THF Ambient 21 %
72
Deoxycholic
acid
Carboxylic
acid/amide
bond
Ethyl chloroformate 24 hours 1, 4
dioxane
Ambient Not
record
ed
73
183
Lithocholic
acid
Carboxylic
acid/amide
bond
Ethyl chloroformate 24 hours THF Ambient N/A
Deoxycholic
acid
Carboxylic
acid/amide
bond
Ethyl chloroformate 24 hours 1, 4
dioxane
Ambient 21 %
74
Lithocholic
acid
Carboxylic
acid/amide
bond
Ethyl chloroformate 24 hours THF Ambient N/A
Deoxycholic
acid
Carboxylic
acid/amide
bond
Ethyl chloroformate 48 hours 1, 4
dioxane
Ambient 25 %
92
184
Lithocholic
acid
Carboxylic
acid/amide
bond
Ethyl chloroformate 48 hours THF Ambient 53 %
84
Deoxycholic
acid
Carboxylic
acid/amide
bond
Ethyl chloroformate 48 hours 1, 4
dioxane
Ambient 68 %
85
Cholic acid Carboxylic
acid/amide
bond
Ethyl chloroformate 48 hours 1, 4
dioxane
Ambient 26.8 %
86
Lithocholic
acid
Carboxylic
acid/amide
bond
Ethyl chloroformate 24 hours THF Ambient 19 %
79
185
Deoxycholic
acid
Carboxylic
acid/amide
bond
Ethyl chloroformate 24 hours 1, 4
dioxane
Ambient 8 %
80
Cholic acid Carboxylic
acid/amide
bond
Ethyl chloroformate 24 hours 1,4
dioxane
Ambient 31 %
81
Lithocholic
acid
Carboxylic
acid/amide
bond
Ethyl chloroformate 24 hours THF Ambient 91.8 %
75
Deoxycholic
acid
Carboxylic
acid/amide
bond
Ethyl chloroformate 24 hours 1,4
dioxane
ambient 49 %
76
186
Lithocholic
acid
Carboxylic
acid/amide
bond
Ethyl chloroformate 24 hours THF ambient N/A
Lithocholic
acid
Carboxylic
acid/amide
bond
Ethyl chloroformate 24 hours THF ambient N/A
Cholic acid Carboxylic
acid/amide
bond
Ethyl chloroformate 24 hours THF ambient 1.9 %
89
187
Scheme 13: Activation of lithocholic acid by means of ethyl chloroformate followed by amide
formation.
As the aminolysis reactions were not very successful, often with multiple side reactions as well as low
yields of desired compound, other coupling methods were investigated, such as DMT-MM and water
soluble carbodiimides. The reaction conditions were varied but the reactions were never successful
and starting material was always recovered. This was possibly to do with either solubility issues as
previously discussed or the possibility that the steroidal moiety was causing some kind of steric
hindrance.
Ethyl chloroformate was the only successful coupling reagent which was found for the production of
bile amides in this particular project. Ethyl chloroformate can also potentially react with the hydroxyls
present on the steroid, producing carbonates. Although it will preferentially react with the carboxylic
acid, if the initial mixed anhydride stage is left for too long a period (over the initial 2 hours) or in an
excess, the carbonate will form (fig 28). This makes purification from these, already sometimes low
yielding reactions even more difficult. The problem was overcome by only adding 0.99 equivalents of
ethyl chloroformate. By only adding 0.99 equivalents, it was guaranteed to have some starting material
leftover. However, it is easier to remove starting acid by washing with saturated sodium hydrogen
188
carbonate solution so this was preferable to formation of the carbonate. It has been reported and found
that these reaction between acids and ethyl chloroformate type reagents are typically very quick and
very exothermic so it was essential to keep the materials cool while the ethyl chloroformate was added
dropwise over 10 minutes, otherwise, non-homogeneous reaction products will be formed (Ulmer,
1999). The usual methodology for this was to keep the reaction vessel on ice, apart from when using 1,
4-dioxane, as the solvent would freeze under these conditions, so cold water was used instead.
Fig 28: Mixed anhydride of lithocholic acid with an unwanted carbonate formation on the 3-OH
position, due to excess ethyl chloroformate (compound 133).
3.8.1 Inverse addition amide formation reactions
Fig 29: Compound 87, an excellent germinator of C. difficile spores.
133
87
189
One compound that was tested as a germinant of C. difficile spores had a free NH2 group present
(compound 87). The results received from this were very promising so more compounds with free
NH2 groups were thought to be the way forward. However, this was a difficult synthesis and wasn’t
successful with either deoxycholic acid or the cholic acid. The problem looks like dimers were formed
(fig 30), even though the anhydride was added slowly in inverse conditions, the formation of dimers
still occurred or there was such a multitude of compounds from side reactions, that purification was
impossible. Although compound 30 was produced by aminolysis with a methyl ester and amine, the
same methodology was applied for the other two bile acids, without the same success. This is one of
the more unusual situations where the deoxycholic derivative is relatively easily synthesised in
comparison to the other two bile acids. Usually, lithocholic and cholic acid derivatives were easier to
synthesise/purify than the deoxycholic acid variants.
Fig 30: Possible dimerization formation from mixed anhydride induced inverse addition reactions
(compound 134).
It was found that the ethyl chloroformate induced anhydride method was a reasonable method for the
synthesis of bile amides on a 0.5 g scale. To produce enough material for the further quaternization of
the material it was decided that a moderate scaling up would be required so as to produce all the
required analogues while being as time efficient as possible. It was decided that a 2 g scale would
suffice and the reagents were scaled up appropriately. However, it was found that if the reagents were
134
190
left in the same molar ratios when scaling up, the reaction would not occur. It was eventually found
that for the scaling up of the material, 4 x the equivalent of the base (usually triethylamine) was
required for the reaction to occur to a satisfactory yield.
191
3.9.1Attachment of pendant groups on 3-OH
Table 6: All reactions attempted to manipulate the 3-OH moiety on lithocholic acid.
Parent bile acid Attachment position/bond
formation
Reagent Base Catalyst Temperature oC
Time Solvent Yiel
d
Methyl lithocholate 3-OH, ether
NaI 90 5 hrs. Acetonitril
e
N/A
Methyl lithocholate 3-OH, ether
NaH NaI ambient 24
hrs.
Acetonitril
e
N/A
Methyl deoxycholate 3-OH, ether
Cu(acac)2 120 24
hours
Vinyl
benzyl
chloride
N/A
Lithocholic acid 3-OH, ester
Ambient 30
mins
THF N/A
Lithocholic acid 3-OH, ester
Ambient 30
mins
Ethylene
glycol
N/A
192
Lithocholic acid 3-OH, ester
DMAP 100 24
hours
THF N/A
Deoxycholic acid 3-OH, ester
100 48
hours
Ethylene
glycol
N/A
Compound 61 3-OH, ester
ambient 4
hours
Chloroform N/A
Lithocholic acid 3-OH, ester
DMAP ambient 12
hours
Chloroform N/A
Methyl lithocholate 3-OH, ester
ambient 24
hours
Chloroform 57 %
100
Compound 93 3-OH, ester
ambient 5
days
chloroform 6.77
%
102
193
3.10.1 Ether linkage
Scheme 14: Attachment of 4-vinyl benzyl chloride to methyl lithocholate.
The ultimate aim of the project was to produce a germinatory, antibacterial polymer bile amide
compound which would be able to form a surface. Different methods were investigated to see which
methodology would be the most effective at achieving this goal. As literature suggested that 3-OH was
less important in the germination of C. difficile this was the position which was used as the target for
polymer formation.
One failed attempt to attach a group to the 3-OH of lithocholic acid was using Cu(acac)2 as a catalyst
to attach an alkyl halide to the hydroxyl group to form an ether bond. Following the literature
(Sirkecioglu et al., 2003) this is where heating of the two starting materials with a catalytic quantity
of Cu(acac)2 was shown to produce the ether bond in high yields. This was tried with vinyl benzyl
chloride and lithocholic acid. In the literature this was successful with benzyl chloride and cholesterol,
which are not that different.
Another attempt to form an ether bond on the 3-OH of the lithocholic acid was through the use of
sodium hydride (Clayden, 2009). Sodium hydride is a very strong base, which is required to remove
the hydrogen from the alcohol group, to produce an alkoxide ion. The vinyl benzyl chloride, which is a
suitable electrophile, then reacts with the alkoxide, producing the ether. This reaction was tried several
times, with differing concentrations of NaH, none of which was successful.
194
3.11.2 Ester linkage
Scheme 15; Formation of an ester linkage at the 3-OH position on methyl lithocholate, using acryloyl
chloride.
To produce a potential polymer by attaching a polymerizable group to the 3-OH several different
attempts were made, including the use of both methacryloyl chloride and methacrylic anhydride,
which were both unsuccessful in relation to the bile acid structure. This was later found to also be true
in the literature (Hu et al., 2005). When acryloylating the 3-OH with acryloyl chloride it was found
that the determining factor as to whether the reaction would work or not was the amount of the
acryloyl chloride relative to the bile acid/amide. There needed to be at least three times the equivalent
amount of the acryloyl chloride for there to be completion of the reaction. Two equivalents was not
enough and with this amount there was still a considerable amount of starting material after the same
time frame had passed, thus identifying the amount as opposed to time being the critical factor in
determining reaction success. No catalyst was used, but some literature suggest this might make a
difference (Hu et al., 2005). Although the reaction time was not that long (~3 days), the catalyst may
be helpful if the product turned into a commercial venture. The reason that acryloyl chloride was used
was because the methacryloyl chloride could not be attached to the 3-OH. This was tried several times
with different concentrations, but was not successful, unlike the acryloyl group.
195
3.12.1 Polymeric compounds.
Fig 31: A potential polymerised co-germinant, based on L-lysine (compound 135).
As co-germinants are an important factor in increasing the likelihood of germination, work was done
on incorporating a glycine analogue (glycine shown to be the most effective co-germinant) and
produce a polymeric version of this material. As it was hoped that introduction of a quaternised
ammonium ion would produce an antimicrobial effect, this was initially attempted by using an
alkylating preformed polymer to produce the material (135).
Boc-N’, N’-dimethyl-L-lysine hydrochloride reacted with poly vinyl benzyl chloride for 24 hours.
Removal of the solvent produced an insoluble white powder. Thus solution phase analysis was
impossible. The fact that the product was completely insoluble yet the starting materials were very
soluble in chloroform, would suggest that a reaction had taken place and it could be postulated that the
material in fig 31 had been made. Since it was insoluble in most solvents, there is the possibility of
crosslinking occurring. One possible type of crosslinking that could have occurred would be via
polymer bound benzyl ether formation. This type of crosslinking usually requires elevated
temperatures and a catalyst however, neither of which were present (Dahan and Portnoy, 2003). More
success was found when working on the polymerizable amino acids towards the end of the project,
although literature (Howerton et al., 2010) suggests that L-lysine was not a good germinant on its own,
by manipulating the side chain something could be produced which might behave like a glycine in
terms of co-germinating, due to the fact the stereochemistry was correct and the amino group and the
135
196
carboxylic acid group were both free, something which again, literature suggests is very important for
activity.
Scheme 16: Compound 101, a methyl lithocholate which has had an acryloyl group attached at the 3-
OH position, which has then been polymerised.
Compound 101 is the polymerised (at 3-OH) methyl ester of lithocholic acid. At room temperature
bile acid acrylates are crystalline and are easily polymerizable (Ahlheim et al., 1986). This material
was shown in the proton NMR spectrum to have polymerised by the loss of the two doublets from the
acryloyl group (at 5.72 and 6.31 ppm respectively) and broadening of all the other steroidal peaks.
What was unusual about this compound is the fact that although there was loss of key peaks and
broadening of the 3-CH which would be expected, there was distinctly less broadening of the methyl
peaks. There are two possible reasons for this, the first being that the polymers were of very short
chain lengths, and therefore there was not much broadening of the methyl peaks. The second reason
may be because the methyl peaks are so far away from the polymerised end, they still have sufficient
mobility to allow relaxation of the 1H nuclei and therefore broadening does not occur.
197
3.13.1 Quaternization of materials to induce antimicrobial activity.
Bile amide Alkylating
agent
solvent Temperature oC time Yield Prod
compound
number
Compound 62
Iodomethane Chloroform ambient 24 hours 81.5 % 103
Compound 62 Ethyl iodide chloroform Ambient 24 hours 76 % 105
Compound 62 Allyl bromide chloroform Ambient 24 hours 54 % 107
Compound 62 Vinyl benzyl
chloride
chloroform Ambient 24 hours 26.9 % 109
198
Compound 63
Iodomethane chloroform Ambient 24 hours 78.9 % 104
Compound 63 Ethyl iodide chloroform Ambient 24 hours 52 % 106
Compound 63 Allyl bromide chloroform Ambient 24 hours 53 % 108
Compound 64
Iodomethane chloroform Ambient 24 hours 64.6 % 110
Compound 64 Iodopentane dichloromethane 40 24 hours 50.7 % 124
Compound 64 Allyl bromide dichloromethane Ambient 24 hours 71 % 112
compound 64 Iodopropane chloroform ambient 96 hours N/A
Compound 64 Iodobutane DCM Ambient 48 hours N/A
199
Compound 64 Iodopentane DCM Ambient 24 hours 50.7 % 124
Compound 64 Iodohexane DCM Ambient 24 hours N/A
Compound 64 1-bromo-3-
phenylpropane
DCM Ambient 48 hours N/A
Compound 64 Cyclopentyl
bromide
Chloroform Ambient ~ N/A
Compound 64 Benzyl
bromide
chloroform Ambient 48 hours 35 % 113
Compound 64 Vinyl benzyl
chloride
chloroform Ambient 48 hours 75 % 114
Compound 64 Poly (vinyl
benzyl
chloride)
chloroform 50 24 hours 20 % 120
Compound 64 Chloromethyl
pivalate
chloroform Ambient 96 hours N/A
200
Compound 65
Iodomethane chloroform ambient 24 hours 75 % 111
Compound 65 Poly (vinyl
benzyl
chloride)
chloroform Ambient 24 hours 81 % 121
Compound 75
Iodobutane Chloroform Ambient 48 hours N/A
Compound 75 Allyl bromide chloroform Ambient 1 week 83 % 107
Compound 75 Chloromethyl
pivalate
dichloromethane Ambient 72 hours N/A
201
Compound 75 Vinyl benzyl
chloride
dichloromethane Ambient 48 hours 12 % 119
Compound 76
iodomethane chloroform Ambient 1 week 66 % 115
Compound 76 Allyl bromide chloroform Ambient 24 hours 27 % 116
Compound 76 iodobutane Chloroform Ambient 48 hours N/A
Compound 76 Benzyl
bromide
dichloromethane ambient 24 hours 1.5 % 118
Allyl bromide dichloromethane Ambient 96 hours 83 % 117
202
Compound 94
Iodomethane chloroform ambient 24 hours 4 % 122
Compound 94 Allyl bromide chloroform ambient 24 hours 8 % 123
Compound 94
Ethyl iodide chloroform Ambient 48 hours N/A
Compound 94 Propyl iodide chloroform Ambient 2 weeks N/A
Compound 94 Butyl iodide chloroform Ambient 48 hours N/A
Compound 94 Iodohexane chloroform Ambient 48 hours N/A
Compound 94 Vinyl benzyl
chloride
chloroform Ambient 24 hours 38 % 125
Table 7: All the alkylation reactions with amide analogues of both lithocholic acid and deoxycholic acid.
203
Scheme 17: Quaternization of a tertiary amine by an alkylating agent.
The purpose of quaternization of the tertiary nitrogen in the compounds was to try and induce some
antimicrobial effects. Although the original idea was to produce a material which would initially
germinate the C. difficile spore then, once it had germinated, the quaternized material would be able to
kill it. What has actually happened is that a potential sporicide has been produced. This has the
potential added advantage of being non-specific so would kill any bacteria which it came into contact
with, which in a hospital setting, is a positive quality.
What was observed, out of a moderate range of differing alkylating agents, that products which had
been quaternized with either iodomethane, allyl bromide or vinyl benzyl chloride, had a tendency to
precipitate out of the chloroform solution in the purest form, in the quickest time (~1-2 days). It is
clearly nothing to do with the counter ion as all three reagents used here precipitated. Iodoethane,
iodobutane, iodopentane and iodohexane would always require a work up for them to be a pure
product and would generally come out in a much smaller yield.
Fig 32: Compound 121, a quaternized polymeric compound of deoxycholic acid.
121
204
Compound 121 (fig 32) is a polymerised, deoxycholic acid quaternized derivative. This compound has
not been tested yet as a germinant. When synthesising this compound there was a 10 x equivalent
excess of poly(vinyl benzyl chloride) 60/40 mixture of 3- and 4- isomers, therefore, even with 100 %
completion, there would have only been 10 % quaternization. Once it had been established that the
material was formed it was suggested that a higher loading should be tried. The reaction was
completed again with 5 x equivalents of the bile amide. Once again the material precipitated out. To
ensure the material was pure, it was washed in hot chloroform (as both starting materials were soluble
in chloroform). Once washed the material was dried under vacuum. It was then discovered that this
material was insoluble in H2O, MeOH, DMSO, THF and toluene, so a proton NMR was not
achievable. The suggestion for the insolubility would be that crosslinking had occurred. From the
inherent structure, the only possible crosslinking that can be suggested is a Williamson ether linkage
whereby the free benzoyl chlorides are reacting with the 3-OH to form an ether, thus crosslinking the
material, thus making it insoluble (Clayden, 2009). The way to confirm or deny this proposition is
through the use of solid state NMR or X-ray diffraction which was not available to the researcher at
the time of the project.
Compound 120 was the lithocholic analogue of compound 121. This was only tried with the higher
loading of bile amide to encourage more bile acid pendant groups. However, like compound 64, there
is the possibility that crosslinking has occurred due to insolubility in DMSO, H2O, TFA, AcOH,
acetone, 1, 4-dioxane, pyridine and 1,2-dimethoxyethane. Again, like compound 121, the only way to
confirm the structure is through solid state, but the fact that the material precipitated out after a night
in the container would indicate that there has been some quaternization occurring, due to the
insolubility of the material in chloroform.
205
Fig 33; Compound 120, a potentially antimicrobial steroidal based polymer.
Fig 34: Compound 72, an aniline derived bile amide.
Compound 72 was synthesised (along with deoxycholic acid analogue) as a potential polymerizable
germinant. Although it is yet to be tested, there was some work on trying to polymerise the vinyl
group attached to the material using AIBN as a free radical initiator. This was not successful and there
was always a significant amount of starting material compared to potential product. Several attempts
were made, usually extending the time of exposure to heat and also of adjustment of the amount of
AIBN in the reaction vessel, all to no avail. This would seemingly suggest that once the amide
functionality is somehow affecting the materials ability to polymerise, or perhaps there are certain
steric constraints due to the steroidal material attached to the vinyl aniline.
120
72
206
3.14.1 Synthesis of amino acid analogues
Parent
amino
acid
Attachment
position/bond
formation
Reagent Reagent 2 Catalyst Temperature oC Time Solvent Yield Compound
number
Boc-
Lys-
OH
Amine/amide
bond
Ambient Methanol N/A
Boc-
Lys-
OH
Amine/amide
bond
DMAP Ambient 24 hours chloroform N/A
Boc-
Lys-
OH
Amine/amide
bond
NaOH Ambient 24 hours 1,4-dioxane N/A
Boc-
Lys-
OH
Amine/amide
bond
Ambient 24 hours 1, 4-
dioxane
N/A
207
Boc-
Ser-
OMe
Hydroxyl/ether
Potassium
carbonate
NaI 40 24 hours Acetonitrile N/A
Z-
Glu-
OMe
Carboxylic
acid/amide
N-(3-
dimethyla
minoprop
yl)-N’-
ethyl
carbodiimi
de
Ambient 24 hours Dichlorome
thane
>20
mg
126
Boc-
Lys-
OH
Amino/ amide
NaOH Ambient 2 hours 50/50
acetonitrile/
2 M NaOH
13 % 127
Table 8: All the reactions attempted to make amino acid derivatives.
208
Once a relatively large potential germinatory database had been acquired, towards the end of the
project, the focus shifted towards the preparation of polymeric amino acids. Howerton et al, 2010
suggest that glycine is the best co-germinant for germination of C. difficile spores so any polymeric
analogue of this material would possibly also have some co-germinating ability.
The initial attempt to join a polymerizable moiety to an amino acid was 4-vinyl aniline attaching to Z-
Glu-OMe via a mixed anhydride. It was observed during the reaction that the material started off
colourless but over a period of 24 hours turned darker and darker till the solution was completely
brown. A work up was done but no desired product obtained.
Scheme 18: Synthesis method for a failed coupling between Z-Glu-OMe and 4-vinyl aniline.
The second methodology was also unsuccessful. This involved the coupling of Boc-Ser-OH with vinyl
benzyl chloride in a Williamson-ether synthesis with potassium carbonate as the base. After work up,
none of the desired product was found. Perhaps the reason for failure was that potassium carbonate
was not a strong enough base. If sodium hydride had been used this may have caused the reaction to
go to completion.
209
Scheme 19: Attempted coupling of Boc-Ser-OH with vinyl benzyl chloride.
The final attempt to attach an amino acid to a polymerizable group was the reaction of Boc-Lys-OH
with methacrylic anhydride. This in-situ reaction unfortunately did not work and none of the desired
compound was recovered.
Scheme 20: Attempted coupling of methacrylic anhydride to Boc-Lys-OH.
Compounds 126 and 127 are examples of successful attachment of polymerizable groups to amino
acids.
210
Scheme 21: Synthesis of manipulated amino acid (126) using a carbodiimide coupling agent.
Scheme 22: Compound 127; a monomeric polymerizable amino acid analogue.
Compound 126 was successfully synthesised from Z-Glu-OMe and 4-vinyl aniline using N-(3-
dimethylaminopropyl) N’-ethyl carbodiimide hydrochloride as the coupling agent. This reagent is
specifically used for formation of amide bonds, but is more usually used for peptide synthesis. This
compound was hard to purify as the proton NMR strongly indicated there was an impurity, which
could not be visualised via TLC. Eventually it was removed through precipitation in water from
methanol. There was an attempt to polymerise the material, but in the short time left, this was not
achieved. Given that the bile amide derivative with vinyl aniline attached didn’t polymerise, then it
may be that vinyl aniline derivatives do not polymerise well.
126
127
211
The synthesis of compound 127 did not require a coupling agent. Acryloyl chloride was used to
produce the amide bond. Although it was a low yield, the material was successfully synthesised. There
was no time to attempt polymerization of this material. As there has been successful polymerization of
acryloyl chlorides with bile acids attached, there may be the possibility of being able to produce a co-
polymer containing a co-germinant with a germinant.
As mentioned in the introduction there is a lot of evidence to back up the idea that glycine is a co-
germinant of C. difficile spores. There was a suggestion of attaching Boc-Lysine-OH to methyl
lithocholate through an aminolysis reaction. This produced a TLC with 6 individual spots on the plate,
so the reaction was discontinued, as the purification would not be worth the small amount of material
recovered. However, this reaction was successful with ethyl chloroformate for both the lithocholic
acid and deoxycholic acid, but not with cholic acid.
Scheme 23: Mixed anhydride method of attaching an amino acid group to a bile acid.
3.15.1 Bile amides containing fluorescent side chains-anthranilamide
Three bile amide analogues containing 2-aminobenzamide were synthesised to see whether, upon
germination testing, staining of the spores or vegetative C. difficile cells could be observed under a
confocal microscope. This could potentially give more insight into the binding mechanism of the
germinants and thus give more data about how germination actually occurs in C. difficile.
The three analogues were derivatives of lithocholic acid, deoxycholic acid and cholic acid. Before
submission for germinating studies (which was undertaken by microbiologists Kristian Poole and
212
Amber Lavender), the materials were placed in a 96 well-plate and scanned using a UV spectrometer.
From this analysis it was found that the optimum excitation limit was 300 nm and the optimum
emission was 464 nm. This result was observed for all three bile amide analogues. If the fluorescent
bile acid analogues were to bind to the spores, but not germinate them, this may give some indicator of
binding sites on the spore surface. If the bile amide analogue did germinate the spores, it could be a
potential germinant. However, under the confocal microscope, after washing, it was found that the
material had not bound to the spores or caused germination. This was the same for all three analogues,
so research in this particular area was discontinued.
Fig 35: A bile amide analogue with 2-aminobenzamide (compound 84).
A similar material (compound 91) also had the presence of the aminobenzamide group and it was
hoped the material would be able to be observed interacting with C. difficile spores in some way. At
the time of writing, no testing on this material had been done.
84
213
3.16.1Benzophenone-based materials.
Scheme 24: Synthesis method for a coupling between lithocholic acid and 4-amino-benzophenone.
Benzophenones, through a process called hydrogen abstraction, form radicals, which have been shown
to chemically attach to any organic material which contains a C-H bond and therefore become
incorporated into the material. Thus an entire surface of benzophenone based material could be
produced by exposing them to UV radiation and reacting with each other and the surface (Scheme 22).
This would be another way of producing a “polymer” like surface and would in theory be a better
option, as the material could be reapplied much more easily. The reason for the synthesis of the
benzophenone based compounds, including the three bile acid derivatives was based on the literature
that exposure of benzophenone moieties to UV radiation initiates hydrogen abstraction. This creates
radicals on the benzophenone structure, which can attach to any C-H group, thus providing a surface
with potential germinating materials on it.
Scheme 25: Binding of benzophenone based bile amides to organic surface containing C-H bonds.
214
The fact that no incorporation into the pre-formed polymer structure occurred, suggests several
possibilities for not working. The first is that the bile amide with benzophenone and the addition of an
amide group next to it, reduced the ability of the benzophenone to undergo hydrogen abstraction, thus
no polymer was formed. The second may be the selection of polymer used, which is unlikely as it was
a benzophenone based polymer, which would make the most sense to be attached to, as they are
inherently attached to each other. Another reason may be length of exposure. The problem with
exposure times is that the environment becomes quite warm and therefore there is a substantial loss of
solvent, which needs to be replaced.
Fig 36; Compounds 129 and 130, used in the potential production of benzophenone based surfaces.
The reason the other two compounds (129 and 130) were synthesised was the possibility of attaching
these materials to other surfaces, then attaching the bile amide version to it, thus having an in-situ
129
130
215
polymer preparation. Due to the lack of hydrogen abstraction on the normal polymer and then the
added knowledge of the lack of germinating ability of the material, this idea was discontinued.
216
4.1.1 Germination results
Compound
number
Compound structure Log
reduction
ice
11204
Log
reduction
ice 027
Log
reduction
heat
11204
Log
reduction
heat 027
Inference
60
0.69 0.92 0.78 0.72 Germinant/potential
antimicrobial/sporicide
65
1.22 1.79 Germinant
222
88
0 0.45 0 0 Negligible germinating
ability.
87
2.62 1.72 3.37 2.91 Good germinant.
Probable
antimicrobial/sporicide.
223
92
0.96 0.27 N/A N/A Germinant.
109
1.32 1.27 2 1.5 Germinant.
Possible
antimicrobial/sporicidal
activity as well.
224
93
0 0 0 0 No activity
116
1.05 N/A 0.71 N/A Germinant of 11204.
Also possible
antimicrobial/sporicidal
activity.
225
124
N/A 1.58 N/A 2.08 Germinant of 027.
Possible
antimicrobial/sporicide
action.
112
0 0 0 0 No germination
activity.
Table 9: Germination results for bile amide derivatives against C. difficile spores.
226
Table 9 gives the log reductions, in both heat and ice of all the compounds which were able to be
tested; in due time it is hoped that this database will be increased. The reason for using the ice and heat
method was to determine potential antimicrobial ability. After testing, the plates were read to see the
number of colonies available. If there was a reduction on the heat, this meant that the compound being
tested had germinated the spores and any vegetative cells destroyed by the heat. If there was a
reduction in colony numbers on ice this meant that the material was either germinating, then killing
the vegetative cells or was simply a sporicide.
Due to the insolubility of certain compounds in certain solvents and the fact that chloroform is not
allowed to be used as a solvent for biological testing, the solvents themselves (40% ethanol, 80 %
DMSO, 100 % DMSO) were run. It was shown that the use of these solvents did not have an effect on
the spores ability to germinate. Before analysis was completed, statistical testing was done on the data,
which showed that there was no significant difference between the two different strains tested.
Therefore, if there is a difference, this is specifically related to the compound which is being tested.
The method used by the microbiologists is as follows:
A 2% (w/v) sodium taurocholate comprising double strength thioglycolate germination solution was
prepared as Wheeldon et al (2008). 200µL was added immediately, in triplicate, to 200µL C. difficile
NCTC 11204 spores from an original stock suspension of ~106cfu/mL in microfuge tubes and
vortexed. After 1 hour exposure, 600µL sterile distilled water was added to the test samples and
vortexed, and then subsequently heated in a water bath to 75°C for 20 minutes. Control samples
contained 200µL spore suspensions and 200µL sodium taurocholate germination solution, after 1 hour
exposure 600µL sterile distilled water was added to samples, vortexed and were subsequently stored
over ice for 20 minutes.
Following heat-shock, serial dilutions were performed and samples were inoculated onto Fastidious
Anaerobic Agar comprising 5% (v/v) defibrinated horse blood and 0.1% (w/v) sodium taurocholate.
Serial dilutions and inoculations were repeated for control samples.
227
Method adapted for alternative germination solution consisting 5mL DMSO and 5mL ‘Tween 20’ in
germination solution comprising 2% (w/v) sodium taurocholate and double strength thioglycolate
medium.
When referring to log reduction of spore counts, 1 log reduction would be equivalent to a 90 %
reduction in the spore count, 2 log reduction would be 99 %, 3 log would be 99.9 % and so on.
Compound 60
From the literature (Howerton et al., 2010) it would be hypothesised that as this material is both a
lithocholic derivative and has a ester bond instead of a amide bond that it would have no C. difficile
spore germinating ability whatsoever. However, while testing with ice, for strains 11204 and 027 the
log reduction in number was 0.69 and 0.92 respectively. This shows that while there is not a huge
amount of germination occurring, this particular analogue is a better germinant than lithocholic acid
on its own. The effect that the ester bond has on the material’s germinating ability may be somewhat
displaced by the presence of the terminal hydroxyl on the end of the ethylene glycol group, which may
help to improve germinating ability. With heat there was a larger log reduction in the strain 11204 than
in strain 027 (0.78 and 0.72 respectively). The fact that there is similar log reduction (and therefore
spore count) in both the heat and the ice would imply there may be some antimicrobial ability, because
the ice itself would not be able to kill any vegetative cells. Whether the material is sporicidal or
antimicrobial is not clear and could not be proved unless testing on another spore forming bacteria
were to be attempted (such as Bacillus subtilis).
228
Compound 65
From the literature (Howerton et al., 2010) it would be hypothesised that as this material is a
deoxycholic acid derivative and has an amide bond that it would have some C. difficile spore
germinating ability. When tested with ice, for strains 11204 (027 not recorded) the log reduction in
number was 1.22. With heat 11204 had a log reduction 1.79. The fact that there is a higher reduction
with ice would imply there is no antimicrobial ability, and this material is purely a germinant. There is
no quaternized nitrogen in this material so there was no expectancy of antimicrobial ability, but as it
had an amide bond and two hydroxyls, some germination was expected. As this has moderate
germinating ability it should be noted that there are three carbons in the functional chain, as later
materials, with differing chain lengths possess different properties.
Compound 81
Out of all the three bile acids being worked on in this project, cholic acid has been consistently
reported in the literature to be the better germinant, compared with both deoxycholic and lithocholic
acid. In the case of strain 11204 this particular cholic acid analogue, the log reduction of the material
229
with regard to germination was 0.38, which is insignificant as a germinant. With the 027 strain there
was no germinatory ability present whatsoever. This shows that clearly the benzophenone side chain
group is a significant inhibitor of the ability of the cholic acid fragment to induce germination.
Compound 106
This material is similar to compound 105 but the difference between them is the fact that this is the
deoxycholic acid version, as opposed to compound 105 which is the lithocholic acid version. The
addition of the extra hydroxyl at position 12 improves the germination properties of this material.
There is no improvement with regards to germination of strain 027 but 11204 has a log reduction
difference of 0.75 on ice and 1.38 on heat. This seems to go along with the current literature, which
specifies that deoxycholic analogues generally, are better than lithocholic acid versions. The fact that
there is an improvement, by the addition of an extra hydroxyl, seems to suggest that the counter ion is
not playing an important part in the germinatory process, or at least, in this case, iodine is not a
problematic counter ion.
230
Compound 82
This compound is the reduced version of compound 79, and while that compound had no germinatory
effects whatsoever, compound 82 has a log reduction on ice of 0.46 in strain 11204 (027 no significant
effect). The only difference between the two compounds is the reduction of the ketone to produce the
new hydroxyl group. This extra hydroxyl has clearly had an effect on the germinatory behaviour, even
if it is a small effect. It would appear that the introduction of hydroxyls into the side chain can have a
positive effect on the promotion of germination. Unfortunately, there was no time to synthesise and
test the other analogues of this material to see if this was just an improvement for the lithocholic acid
or whether it would be across the entire analogue series.
Compound 88
This material only showed log reduction of germination in one of the strains which was tested on. In
strain 027 there was a log reduction of 0.45, whereas in 11024 there was no reduction. The fact that
231
this is the lithocholic version means this would be the least likely to be a germinant, however, it would
be a good idea to test the cholic acid version (which has been synthesised), to see if there was any
improvement in the germinating ability. It may be that the side chain is too long and flexible so may
not be able to interact with the active site, but free NH2 groups seem to have a positive effect on the
germination ability, much like free OH on the side chain do, but the lack of hydroxyls on the steroid
structure may be limiting the germinating ability.
Compound 87
Compound 87 was the most successful germinant produced in this project. On ice for strain 11204 it
has a log reduction of 2.62 and for 027 it has a log reduction of 1.72. On heat the log reduction is even
higher with strain 11204 having a log reduction of 3.37 and for 027 the log reduction is 2.91. So while
this product is clearly an excellent germinant of C. difficile spores the large log reduction even on ice,
would imply that there is some antimicrobial/sporicidal activity also taking place as there is a large log
reduction. The fact that this compound is a deoxycholic analogue ties in with the literature on this
compound as a germinant. It is an amide and, similar to earlier compounds with 2 carbons in their side
chain (which had no germinatory properties in other compounds). Perhaps the most important factor
though is the presence of the free NH2 terminal group which is similar to compound 31 (which was
able to induce some germination). It would appear from this data that the 7 hydroxyl is not essential
for germination but that a free amine is. If the analogues were to be synthesised and tested, this may
provide more information about the particular importance of hydroxyl positions on this type of
compound. The analogues of this compound (lithocholic and cholic) were attempted but proved to be
problematic in their syntheses and purification. As the testing wasn’t done immediately, the
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importance of these molecules was missed and therefore any future work should definitely involve
these analogues as a priority.
This particular compound is not dissimilar to the compound 57 Lithocholyl-N-2-(2-aminoethyl)amide,
which is a proven antimicrobial (Ahonen et al., 2010). The key similarities are the steroid backbone,
the amide bond and the free amine group at the end of the side chain. This particular side group may
have antimicrobial properties which need to be fully investigated.
Compound 92
This compound had a log reduction of 0.96 and 0.27 for strains 11204 and 027 respectively. This
structure is very similar to compound 71 except for the addition of a methyl ketone, which, evidently,
has increased the germinating ability to a small level from no activity. This would imply that the
suggestion of the benzene ring being too large to perhaps fit in the binding site was wrong, as there is
no other difference between the two compounds. Perhaps the ketone entity, allows for a greater
amount of hydrogen bonding, similarly to the free hydroxyl groups, found on other germinating
compounds, and it is this hydrogen bonding ability which has increased its germinating ability.
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Compound 109
This compound had a log reduction on ice of 1.32 and 1.27 for strains 11204 and 027 respectively.
The compound had a log reduction on heat of 2 and 1.5 for strains 11204 and 027 respectively. Due to
the fact that there is a large reduction in the ice, coupled with the fact that this material is quaternized,
there could be a possibility of this material exhibiting some antimicrobial/sporicidal effects. It cannot
be known from the data which is provided whether this material is a germinant-antimicrobial or
simply a sporicide. However, testing the material on a bacteria such as bacillus subtilis (a spore
forming bacteria), would mean that this ambiguity could be resolved. Due to the fact that this is very
similar to compound 107, with the only difference being the insertion of a benzene ring into the
alkylating agent (allyl bromide to 4 vinyl benzyl chloride), and the fact that compound 107 had no
germinating ability whatsoever, would imply that this material is a sporicidal agent. Due to the fact
that the inherent steroidal structure is clearly not having any antimicrobial effect, and the literature
suggesting that C. difficile spores are particularly susceptible to chlorine based antimicrobials, it would
appear this vinyl benzyl chloride moiety is of particular interest.
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Compound 116
This compound displays germinating ability on ice for the 11204 strain, with a log reduction in
germination of 1.05 (no germination for 027). It displays a log reduction for the same strain with heat
of 0.71. The fact that there is more reduction over ice than heat would suggest, once again, that this
compound without the allyl ammonium salt may be a germinant/antimicrobial or a sporicide. The
original, un-alkylated version of this compound has not been tested as of yet, but once it has, this will
give a clearer understanding as to whether the material is a germinant/antimicrobial or a sporicide. As
this material is based on lithocholic acid, the presumption would be at this point that this material is
simply a sporicide, due to the lack of lithocholic acid derivatives which have been able to initiate
germination in C. difficile spores. Presuming it is a sporicide, perhaps it is the fact that the counter ion
which is bromine as opposed to chlorine which is limiting its sporicidal activity. If it was possible to
alkylate this material with the same alkylating group, but differing counter ion, this would give some
insight into the importance of the counter ion. The use of ion exchange resins would be able to do this
and thus a fuller investigation of the counter ion could be achieved.
235
Compound 124
This compound had a log reduction on ice of 1.58 for strain 027 and 2.08 for heat (no data for 11204).
The fact that the compound has produced such a relatively large reduction on ice would imply there is
some germinant/antimicrobial or sporicidal activity. This activity could only be eluted if the un-
alkylated material was tested or the compound was tested on other spore forming bacteria such as
Bacillus subtilis. The fact that this material is a derivative of the non-germinating bile acid lithocholic
acid would suggest some potential sporicidal activity, but more testing would have to be done to be
more conclusive.
4.1.2 Overall trends in germination
What the data presented above shows is that the lithocholic amide derivatives are not germinants. It
would therefore be better in future to focus on both deoxycholic and cholic acid as potential
germinating materials. There appears to be an advantage of having some moiety in the side chain
which can become involved in hydrogen bonding (OH and NH). For a more conclusive picture, all of
the compounds synthesised should be tested, then more details on specific needs for germination can
be explored and refined. To confirm whether the material is only a germinant or whether it has
antimicrobial/sporicidal activity then work needs to be done on the compounds using Bacillus subtilis.
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5.1.1 Future work:
Increasing the number of bile amides for screening would ensure that a more complete structure
activity relationship database is produced, as well as continue screening the compounds which have
already been synthesised. This increased database should give more information for what features are
the most optimal for inducing germination and if quaternized, what alkylating agent produces the best
material for antimicrobial activity. Some of the materials already synthesised have been shown to have
potential antimicrobial/sporicidal activity, so testing these materials further on other spore forming
bacteria such as Bacillus subtilis, would enhance the knowledge on whether some sporicidal agents
had been produced. From the germination results so far, backed up by literature, it would be suggested
to concentrate more on deoxycholic and cholic acid as potential germinants, as there doesn’t appear to
be any data to back up the use of lithocholic acid as a germinant of C .difficile.
Due to the very good germination result of compound 87, there is a strong argument for more
analogues of this type, having a free primary amino group. Although the synthesis of these materials
has been difficult, a way around this problem would be to selectively protect one end of the diamine,
then use the anhydride method to attach, and then de-protect to produce the free NH2 group. Using
different length chains would perhaps uncover some more interesting information.
Some of the latter work on this project focussed on the production of polymeric bile acid materials.
There is a lot more work to be done, but since attachment of a polymerizable group has been relatively
easy on the 3-OH (using acryloyl chloride), development of this method should be done. By increasing
the number of potential polymerizable functionalities will be able to see if there are any effects on the
germinatory ability of the material.
Further work should also involve the testing of the co-germinating materials, in conjunction with
known germinatory materials, to see if they can improve germinating ability. Further work should also
be done on trying to produce more polymerizable amino acid analogues, to enhance and optimise co-
germinating ability using the known literature data about co-germinants.
237
Unfortunately there were experiments which were unsuccessful, most notably the benzophenone
analogue work. All the literature seemed to indicate that this would work, however, after several
different attempts, with altering of the conditions, no successful methodology was found. Whether in
future there could be a success by manipulating the benzophenone in a different way to induce
hydrogen abstraction and thus attachment onto a polymeric surface is yet to be seen. There have been
many syntheses of very similar attachments, so there must be something fundamental as to why it
won’t work when attaching the benzophenone to the bile acid.
6.1.1 Conclusions:
Bile amides have been synthesised in the literature before using several different methodologies. What
this project has done, is to optimise the bile amide production using ethyl chloroformate to produce the
anhydride, on a 0.5 g scale and then on a 2 g scale.
One of the aims of this project was to produce a structure activity database with regards to germination
of C. difficile spores. This database has been produced chemically, however, not all of the biological
results are available, and therefore a clearer picture of what is important for germination has not been
achieved yet.
In terms of polymerizable bile acids and bile amides, this has been achieved after several different
methodology attempts were made. There has also potentially been a quaternized, polymeric bile
amide, but due to solubility issues, this cannot be confirmed.
The work on production of polymerizable amino acids, for the specific purpose of use as co-
germinants has not been described in the literature. This work could have a very important role in the
future development of a treatment, should the biological testing come back positively.
While work will continue on a potential polymeric germinating/antibacterial surface there are several
precautions that hospitals could make in the meantime. Enhanced infection control methods would
238
ensure that if there was to be an outbreak, that its chance of spreading to other patients would be
severely limited.
As a separate note, it could be suggested that although this work is valid in the laboratory setting,
under extremely rigid controls; in the real world there may be problems encountered which the
biological experimental protocol cannot account for. One of the limiting factors is that when potential
germinants are being tested, this is in an anaerobic environment and therefore, is not a true reflection
of what will occur in the real life situation. The only way around this is to potentially develop a new
protocol which is more similar to an in vivo situation and thus this will give a more accurate reflection
of the true germinating nature of the compounds produced.
Due to time limits there are tests which should be done before any commercialisation of the materials
occurs. Although a compound may show fantastic properties; great germinant and antimicrobial, this
compound could also be extremely toxic. This would need to be investigated as it might have
unwanted side effects on people who are continuously exposed to it. Also, the surface would
eventually start to break down and would need to be replaced, but it could be potentially quite difficult
to monitor if and when that occurs.
An added advantage to this potentially antimicrobial polymer compound is that it would not be
specific. The quaternary nitrogen ion is active against a broad range of bacteria and it is unlikely to
stay specific to the C. difficile. The potential problem is that it may eventually lead to resistance of
many different types of bacteria which could pose a real threat to the safety of the human species. The
ideal situation would be that there is no antimicrobial activity associated with the compound and that,
with a germinatory material and a stringent cleaning regime; the cell life cycle could be broken.
239
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